Biomembranes@ICMol
List of Publications, including PhD Thesis and main Conferences. Updated:

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    AuthorsTitleYearPublication DataTypeLinks
    Gimenez, D., Sánchez-Muñoz, O.L. & Salgado, J. Direct observation of nanometer-scale pores of melittin in supported lipid monolayers. 2015 Langmuir
    Vol. 31. 3146–3158 
    Article DOI
    Supplement
    Citations 
    Abstract: Melittin is the most studied membrane-active peptide and archetype within a large and diverse group of pore formers. However, the molecular characteristics of melittin pores remain largely unknown. Herein, we show by atomic force microscopy (AFM) that lipid monolayers in the presence of melittin are decorated with numerous regularly shaped circular pores that can be distinguished from nonspecific monolayer defects. The specificity of these pores is reinforced through a statistical evaluation of depressions found in Langmuir-Blodgett monolayers in the presence and absence of melittin, which eventually allows characterization of the melittin-induced pores at a quantitative low-resolution level. We observed that the large majority of pores exhibit near-circular symmetry and a Gaussian distribution in size, with a mean diameter of 8.7 nm. A distinctive feature is a ring of material found around the pores, made by, on average, three positive peaks, with a height over the level of the lipidic background of 0.23 nm. This protruding rim is most likely due to the presence of melittin near the pore border. Although the current resolution of the AFM images in the {x, y} plane does not allow distinction of the specific organization of the peptide molecules, these results provide an unprecedented view of melittin pores formed in lipidic interfaces and open new perspectives for future structural investigations of these and other pore-forming peptides and proteins using supported monolayers.
    BibTeX:
    @article{Gimenez2015,
      author = {Diana Giménez and Orlando L Sánchez Mu&ntil;oz and Jesú Salgado},
      title = {Direct observation of nanometer-scale pores of melittin in supported lipid monolayers},
      journal = {Langmuir},
      year = {2015},
      volume = {31},
      number = { },
      pages = {3146–3158},
      doi = {http://dx.doi.org/10.1021/la504293q}
    }
    
    Aguilera-Mendoza, L., Marrero-Ponce, Y., Tellez-Ibarra, R., Llorente-Quesada, M.T., Salgado, J., Barigye, S.J. & Liu, J. Overlap and diversity in antimicrobial peptide databases: compiling a non-redundant set of sequences. 2015 Bioinformatics
    Vol. 31, pp. 2553–2559 
    Article DOI
    Citations 
    Abstract: Motivation: The large variety of antimicrobial peptide (AMP) databases developed to date are characterized by a substantial overlap of data and similarity of sequences. Our goals are to analyze the levels of redundancy for all available AMP databases and use this information to build a new non-redundant sequence database. For this purpose, a new software tool is introduced. Results: A comparative study of 25 AMP databases reveals the overlap and diversity among them and the internal diversity within each database. The overlap analysis shows that only one database (Peptaibol) contains exclusive data, not present in any other, whereas all sequences in the LAMP_Patent database are included in CAMP_Patent. However, the majority of databases have their own set of unique sequences, as well as some overlap with other databases. The complete set of non-duplicate sequences comprises 16 990 cases, which is almost half of the total number of reported peptides. On the other hand, the diversity analysis identifies the most and least diverse databases and proves that all databases exhibit some level of redundancy. Finally, we present a new parallel-free software, named Dover Analyzer, developed to compute the overlap and diversity between any number of databases and compile a set of non-redundant sequences. These results are useful for selecting or building a suitable representative set of AMPs, according to specific needs.
    BibTeX:
    @article{Aguilera-Men2015,
    Author = {Aguilera-Mendoza, Longendri and Marrero-Ponce, Yovani and Tellez-Ibarra,
       Roberto and Llorente-Quesada, Monica T. and Salgado, Jesus and Barigye,
       Stephen J. and Liu, Jun},
    Title = {{Overlap and diversity in antimicrobial peptide databases: compiling a
       non-redundant set of sequences}},
    Journal = {{BIOINFORMATICS}},
    Journal-ISO = {{Bioinformatics}},
    Year = {{2015}},
    Volume = {{31}},
    Number = {{15}},
    Pages = {{2553-2559}},
    DOI = {{10.1093/bioinformatics/btv180}},
    }
    
    Ruiz-Blanco, Y.B., Marrero-Ponce, Y., Prieto, P.J., Salgado, J., Garcia, Y. & Sotomayor-Torres, C.M. A Hooke's law-based approach to protein folding rate. 2015 J. Theor. Biol.
    Vol. 364, pp. 407–417 
    Article DOI
    Citations 
    Abstract: Kinetics is a key aspect of the renowned protein folding problem. Here, we propose a comprehensive approach to folding kinetics where a polypeptide chain is assumed to behave as an elastic material described by the Hooke's law. A novel parameter called elastic-folding constant results from our model and is suggested to distinguish between protein with two-state and multi-state folding pathways. A contact-free descriptor, named folding degree, is introduced as a suitable structural feature to study protein-folding kinetics. This approach generalizes the observed correlations between varieties of structural descriptors with the folding rate constant. Additionally several comparisons among structural classes and folding mechanisms were carried out showing the good performance of our model with proteins of different types. The present model constitutes a simple rationale for the structural and energetic factors involved in protein folding kinetics.
    BibTeX:
    @article{Ruiz-B2015,
    Author = {Ruiz-Blanco, Yasser B. and Marrero-Ponce, Yovani and Prieto, Pablo J.
       and Salgado, Jesus and Garcia, Yamila and Sotomayor-Torres, Clivia M.},
    Title = {{A Hooke's law-based approach to protein folding rate}},
    Journal = {{JOURNAL OF THEORETICAL BIOLOGY}},
    Journal-ISO = {{J. Theor. Biol.}},
    Year = {{2015}},
    Volume = {{364}},
    Pages = {{407-417}},
    Type = {{Article}},
    DOI = {{10.1016/j.jtbi.2014.09.002}},
    }
    
    Afonin, S., Glaser, R.W., Sachse, C., Salgado, J., Wadhwani, P. & Ulrich, A.S. 19 F-NMR screening of unrelated antimicrobial peptides shows that membrane interactions are largely governed by lipids. 2014 BBA-Biomembranes
    Vol. 1838, pp. 2260–2268 
    Article DOI
    Citations 
    Abstract: Many amphiphilic antimicrobial peptides permeabilize bacterial membranes via successive steps of binding, re-alignment and/or oligomerization. Here, we have systematically compared the lipid interactions of two structurally unrelated peptides: the cyclic β-pleated gramicidin S (GS), and the α-helical PGLa. 19 F-NMR was used to screen their molecular alignment in various model membranes over a wide range of temperatures. Both peptides were found to respond to the phase state and composition of these different samples in a similar way. In phosphatidylcholines, both peptides first bind to the bilayer surface. Above a certain threshold concentration they can re-align and immerse more deeply into the hydrophobic core, which presumably involves oligomerization. Re-alignment is most favorable around the lipid chain melting temperature, and also promoted by decreasing bilayer thickness. The presence of anionic lipids has no influence in fluid membranes, but in the gel phase the alignment states are more complex. Unsaturated acyl chains and other lipids with intrinsic negative curvature prevent re-alignment, hence GS and PGLa do not insert into mixtures resembling bacterial membranes, nor into bacterial lipid extracts. Cholesterol, which is present in high concentrations in animal membranes, even leads to an expulsion of the peptides from the bilayer and prevents their binding altogether. However, a very low cholesterol content of 10% was found to promote binding and re-alignment of both peptides. Overall, these findings show that the ability of amphiphilic peptides to re-align and immerse into a membrane is determined by the physico-chemical properties of the lipids, such as spontaneous curvature. The remarkably similar behavior observed here for two structurally unrelated molecules (with different conformation, size, shape, charge) suggests that their activity at the membrane level is largely governed by the properties of the constituent lipids, while the selectivity towards different cell types is additionally ruled by electrostatic attraction between peptide and cell surface. This article is part of a Special Issue entitled: Interfacially active peptides and proteins.
    BibTeX:
    @article{Afonin2014,
      author = {Sergii Afonin and Ralf W Glaser and Carsten Sachse and Jesú Salgado and Parvesh Wadhwani and Anne S Ulrich},
      title = {19 F-NMR screening of unrelated antimicrobial peptides shows that membrane interactions are largely governed by lipids},
      journal = {BBA-Biomembranes},
      year = {1838},
      volume = {in press},
      number = { },
      pages = {2260–2268},
      doi = {http://dx.doi.org/10.1016/j.bbamem.2014.03.017}
    }
    
    Ruiz-Blanco, Y.B., Marrero-Ponce, Y., Paz, W., García, Y. & Salgado, J. Global stability of protein folding from an empirical free energy function. 2013 J. Theor. Biol.
    Vol. 321, pp. 44–53 
    Article DOI
    Citations 
    Abstract: The principles governing protein folding stand as one of the biggest challenges of Biophysics. Modeling the global stability of proteins and predicting their tertiary structure are hard tasks, due in part to the variety and large number of forces involved and the difficulties to describe them with sufficient accuracy. We have developed a fast, physics-based empirical potential, intended to be used in global structure prediction methods. This model considers four main contributions: Two entropic factors, the hydrophobic effect and configurational entropy, and two terms resulting from a decomposition of close-packing interactions, namely the balance of the dispersive interactions of folded and unfolded states and electrostatic interactions between residues. The parameters of the model were fixed from a protein data set whose unfolding free energy has been measured at the “standard” experimental conditions proposed by Maxwell et al. (2005) and a large data set of 1151 monomeric proteins obtained from the PDB. A blind test with proteins taken from ProTherm database, at similar experimental conditions, was carried out. We found a good correlation with the test data set, proving the effectiveness of our model for predicting protein folding free energies in considered standard conditions. Such a prediction compares favorably against estimations made with FoldX’s function and the force field GROMOS96. This model constitutes a valuable tool for the fast evaluation of protein structure stability in 3D structure prediction methods.
    BibTeX:
    @article{Ruiz-B2013,
      author = {Yasser Ruiz-Blanco and Yovani Marrero-Ponce and Waldo Paz and Yamila García and Jesús Salgado},
      title = {Global stability of protein folding from an empirical free energy function},
      journal = {J. Theor. Biol.},
      year = {2013},
      volume = {321},
      number = {},
      pages = {44–53},
      doi = {http://dx.doi.org/10.1016/j.jtbi.2012.12.023}
    }
    
    Sánchez-Muñoz, O.L., Strandberg, E., Esteban-Martín, S., Grage, S.L., Ulrich, A.S. & Salgado, J. Canonical azimuthal rotations and flanking residues constrain the orientation of transmembrane helices. 2013 Biophys. J.
    Vol. 104(7), pp. 1508–1516 
    Article DOI
    Citations 
    Abstract: In biological membranes the alignment of embedded proteins provides crucial structural information. The transmembrane (TM) parts have well-defined secondary structures, in most cases α-helices and their orientation is given by a tilt angle and an azimuthal rotation angle around the main axis. The tilt angle is readily visualized and has been found to be functionally relevant. However, there exist no general concepts on the corresponding azimuthal rotation. Here, we show that TM helices prefer discrete rotation angles. They arise from a combination of intrinsic properties of the helix geometry plus the influence of the position and type of flanking residues at both ends of the hydrophobic core. The helical geometry gives rise to canonical azimuthal angles for which the side chains of residues from the two ends of the TM helix tend to have maximum or minimum immersion within the membrane. This affects the preferential position of residues that fall near hydrophobic/polar interfaces of the membrane, depending on their hydrophobicity and capacity to form specific anchoring interactions. On this basis, we can explain the orientation and dynamics of TM helices and make accurate predictions, which correspond well to the experimental values of several model peptides (including dimers), and TM segments of polytopic membrane proteins.
    BibTeX:
    @article{Sanchez-M2013,
      author = {Orlando L. Sánchez-Muñoz and Erik Strandberg and Santi Esteban-Martín and Stephan L. Grage and Anne S. Ulrich and Jesús Salgado},
      title = {Canonical azimuthal rotations and flanking residues constrain the orientation of transmembrane helices},
      journal = {Biophys. J.},
      year = {2013},
      volume = {104},
      number = {7},
      pages = {1508–1516},
      doi = {http://dx.doi.org/10.1016/j.bpj.2013.02.030}
    }
    
    Valero, J. G., Cornut-Thibaut, A., Jugé, R., Debaud, A.-L., Giménez, D., Gillet, G., Bonnefoy-Bérard, N., Salgado, J., Salles, G., Aouacheria, A., & Kucharczak, J. µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1) into a prodeath factor reveals two distinct alpha-Helices inducing mitochondria-mediated apoptosis. 2012 PLoS ONE
    Vol. 7(6), pp. e38620 
    Article DOI
    Supplement
    Citations 
    Abstract: Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.
    BibTeX:
    @article{Valero2012,
      author = {Juan García Valero and Aurélie Cornut-Thibaut and Romain Jugé and Anne-Laure Debaud and Diana Giménez and Germain Gilletand Nathalie Bonnefoy-Bérard and Jesús Salgado and Gilles Salles and Abdel Aouacheria and Jérôme Kucharczak},
      title = {µ-Calpain Conversion of Antiapoptotic Bfl-1 (BCL2A1) into a Prodeath Factor Reveals Two Distinct alpha-Helices Inducing Mitochondria-Mediated Apoptosis},
      journal = {PLoS ONE},
      year = {2012},
      volume = {7},
      number = {6},
      pages = {e38620},
      doi = {http://dx.doi.org/10.1371/journal.pone.0038620}
    }
    
    Grage, S. L., Strandberg, E., Wadhwani, P., Esteban-Martín, S., Salgado, J., & Ulrich, A. S. Comparative analysis of the orientation of transmembrane peptides using solid-state 2H- and 15N-NMR: mobility matters. 2012 Eur. Biophys. J.
    Vol. 41(5), pp. 475–482 
    Article DOI
    Citations 
    Abstract: Many solid-state nuclear magnetic resonance (NMR) approaches for membrane proteins rely on orientation-dependent parameters, from which the alignment of peptide segments in the lipid bilayer can be calculated. Molecules embedded in liquid-crystalline membranes, such as monomeric helices, are highly mobile, leading to partial averaging of the measured NMR parameters. These dynamic effects need to be taken into account to avoid misinterpretation of NMR data. Here, we compare two common NMR approaches: (2)H-NMR quadrupolar waves, and separated local field (15)N-(1)H polarization inversion spin exchange at magic angle (PISEMA) spectra, in order to identify their strengths and drawbacks for correctly determining the orientation and mobility of α-helical transmembrane peptides. We first analyzed the model peptide WLP23 in oriented dimyristoylphosphatidylcholine (DMPC) membranes and then contrasted it with published data on GWALP23 in dilauroylphosphatidylcholine (DLPC). We only obtained consistent tilt angles from the two methods when taking dynamics into account. Interestingly, the two related peptides differ fundamentally in their mobility. Although both helices adopt the same tilt in their respective bilayers (~20°), WLP23 undergoes extensive fluctuations in its azimuthal rotation angle, whereas GWALP23 is much less dynamic. Both alternative NMR methods are suitable for characterizing orientation and dynamics, yet they can be optimally used to address different aspects. PISEMA spectra immediately reveal the presence of large-amplitude rotational fluctuations, which are not directly seen by (2)H-NMR. On the other hand, PISEMA was unable to define the azimuthal rotation angle in the case of the highly dynamic WLP23, though the helix tilt could still be determined, irrespective of any dynamics parameters.
    BibTeX:
    @article{Grage2012,
      author = {Stephan L. Grage and Erik Strandberg and Parvesh Wadhwani and Santi Esteban-Martín and and Jesús Salgado and Anne S. Ulrich},
      title = {Comparative analysis of the orientation of transmembrane peptides using solid-state (2)H- and (15)N-NMR: mobility matters},
      journal = {Eur. Biophys. J.},
      year = {2012},
      volume = {41},
      number = {5},
      pages = {475–482},
      doi = {http://dx.doi.org/10.1007/s00249-012-0801-0}
    }
    
    Strandberg, E., Esteban-Martín, S., Ulrich, A. S. & Salgado, J. Hydrophobic mismatch of mobile transmembrane helices: Merging theory and experiments. 2012 BBA-Biomembranes
    Vol. 1818(5), pp. 1242–1249 
    Article DOI
    Citations 
    Abstract: Hydrophobic mismatch still represents a puzzle for transmembrane peptides, despite the apparent simplicity of this concept and its demonstrated validity in natural membranes. Using a wealth of available experimental 2H NMR data, we provide here a comprehensive explanation of the orientation and dynamics of model peptides in lipid bilayers, which shows how they can adapt to membranes of different thickness. The orientational adjustment of transmembrane α-helices can be understood as the result of a competition between the thermodynamically unfavorable lipid repacking associated with peptide tilting and the optimization of peptide/membrane hydrophobic coupling. In the positive mismatch regime (long-peptide/thin-membrane) the helices adapt mainly via changing their tilt angle, as expected from simple geometrical predictions. However, the adaptation mechanism varies with the peptide sequence in the flanking regions, suggesting additional effects that modulate hydrophobic coupling. These originate from re-adjustments of the peptide hydrophobic length and they depend on the hydrophobicity of the flanking region, the strength of interfacial anchoring, the structural flexibility of anchoring side-chains and the presence of alternative anchoring residues.
    BibTeX:
    @article{Strandberg2012,
      author = {Erik Strandberg and Santi Esteban-Martín and Anne S. Ulrich and Jesús Salgado},
      title = {Hydrophobic mismatch of mobile transmembrane helices: Merging theory and experiments.},
      journal = {BBA-Biomembranes},
      year = {2012},
      volume = {1818},
      number = {5},
      pages = {1242–1249},
      doi = {http://dx.doi.org/10.1016/j.bbamem.2012.01.023}
    }
    
    Salgado, J., Giménez, D., Fuetes, G., Cunill, E. & Sánchez-Muñoz, O.L. Lessons from reductionism on a membrane-active protein: minimal pore-forming versions of Bax. 2012 FEBS J.  
    Vol. 279, 22nd IUBMB / 37th FEBS Congress, Sept. 4-9 2012, Seville, Spain pp. 123-124 
    Conference DOI 
    Abstract:

    The proteins of the Bcl-2 family are key regulators of physiological cell death in mammals which control the release of apoptogenic factors from mitochondria. Head members of the family are proapoptotic Bax and Bid and antiapoptotic Bcl-xL. These proteins are amphitropic, as they use dual water-soluble and membrane-bound folds as a strategy for regulation, being the active species membrane-inserted. It is widely accepted that the apoptotic chain of events includes formation of oligomeric pores by Bax in the outer mitochondrial membrane, which is prompted by Bid and can be inhibited by Bcl-xL. However, detailed structural information is only available for the water-soluble species of these proteins, while for the membrane-associated active forms we only have indirect and incomplete information. We are perusing the study of the action mechanism of Bcl-2 proteins in membranes using a reductionist approach which involves the synthesis and characterization of peptides derived from the paradigmatic members of the Bcl-2 family. Peptides encompassing the sequence of either helices from the central hairpin of Bax, Bid and Bcl-xL exhibit membrane binding and activity which correlate with those of their respective proteins of origin. Bax peptide fragments are particularly potent for membrane poration, and in practice behave as minimal versions of the parent protein in synthetic vesicles as well as in isolated mitochondria and living cells. A detailed study of their mechanism of action, including modeling and quantification of the action kinetics, allows defining the type and properties of the pores formed. This work establishes mechanistic links between the action mode of proteins and peptides as pore inducers. In parallel, serves as the basis for the design of a new family of small peptide molecules with strong potential as therapeutic agents.

    BibTeX:
    @conference{Salgado2012,
      author = {jesús Salgado and Diana Giménez and Gustavo Fuetes and Edel Cunill and Orlando L. Sánchez-Muñoz},
      title = {Lessons from reductionism on a membrane-active protein: minimal pore-forming versions of Bax},
      journal = {FEBS J.},
      year = {2012},
      volume = {279},
      number = {S1},
      pages = {123--124},
      url = {http://onlinelibrary.wiley.com/doi/10.1111/febs.2012.279.issue-s1/issuetoc}
      doi = {http://dx.doi.org/10.1111/j.1742-4658.2010.08705.x}
    }
    
    González-Peña, R.J., Sánchez-Muñoz, O.L., Martínez-Celorio, R.A., Cibrián, R.M. Salvador-Palmer, R. & Salgado, J. ζ-potential determination using a ZetaMeter-Dynamic Speckle assembly. 2012 Proc. SPIE  
    Vol. 8413, 5th International Conference on Speckle Metrology, Sep. 10–12 2012, Vigo, Spain p. 84131H 
    Conference DOI 
    Abstract:

    Electrophoretic mobility and zeta-potential are important physical parameters for the characterization of micro-and nano-systems. In this communication we describe a new method for determining the zeta-potential through the assembly of two well known techniques: free electrophoresis and Dynamic Speckle. When coherent light passes through a fluid having scattering centres, the far field interference originates a speckled image. If the scattering centres are contained within the cylindrical electrophoresis cell of a ZetaMeter and are forced to move in an orderly way under the action of an external electric field, the time variation of the light intensity in the far field speckle images follows a temporal autocorrelation function g(tau). The corresponding correlation time can then be obtained and related with the velocity, from which the electrophoretic mobility and the zeta-potential of the scattering centres can be determined. We have applied this method to microparticles, like natural air-floated silica and two classes of bioceramics, hydroxyapatite and biphasic calcium phosphate. For comparison, we analysed the same samples in parallel using a commercial Zetasizer Nano from Malvern Instruments. The values of zeta-potential determined using the two techniques were the same within similar to 3% error. These results validate our new method as a useful and efficient alternative for zeta-potential determination of particles, at least within the micrometer scale.

    BibTeX:
    @conference{Gonzalez-P2012,
      author = {Rolando J. González-Peña and Orlando L. Sánchez-Muñoz and Rene A. Martínez-Celorio and Rosa M. Cibrián and Rosario Salvador-Palmer and Salgado, J.},
      title = {Zeta-potential determination using a ZetaMeter-Dynamic Speckle assembly},
      journal = {Proc. SPIE},
      year = {2012},
      volume = {8413},
      number = {},
      pages = {84131H},
      doi = {http://dx.doi.org/10.1117/12.977785}
    }
    
    Afonin, S., Glaser, R.W., Sachse, C., Salgado, J., Wadhwani, P., Koch, K., Ieronimo, M. & Ulrich, A.S. Lipid composition is the dominant factor that determines the orientational states of membrane-bound antimicrobial peptides. 2011 Eur. Biophys. J.  
    Vol. 40 S1, 8th EBSA European Biophysics Congress, Aug. 23–27 2011, Budapest, Hungary, p. 194 
    Conference
     
    Abstract:

    BibTeX:
    @conference{Afonin2011,
      author = {Afonin, S., Glaser, R.W., Sachse, C., Salgado, J., Wadhwani, P., Koch, K., Ieronimo, M. & Ulrich, A.S.},
      title = {Lipid composition is the dominant factor that determines the orientational states of membrane-bound antimicrobial peptides},
      journal = {Eur. Biophys. J.},
      year = {2011},
      volume = {40},
      number = {S1},
      pages = {194},
      doi = {}
    }
    
    Valero, J. G., Sancey, L., Kucharczak, J., Guillemin, Y., Giménez, D., Prudent, J., Gillet, G., Salgado, J., Coll, J.L., & Aouacheria, A. Bax-derived membrane-active peptides act as potent and direct inducers of apoptosis in cancer cells 2011 J. Cell Sci.  
    Vol. 124, pp. 556-564 
    Article DOI
    Supplement
    Citations  
    Abstract: Although many cancer cells are primed for apoptosis, they usually develop resistance to cell death at several levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members such as Bax, is considered as a point of no return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on minimal active versions of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, which can target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax- derived ‘poropeptide’ produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins that form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.
    BibTeX:
    @article{Valero2011,
      author = {Juan García Valero and Lucie Sancey and Jèrôme Kucharczak and Yannis Guillemin and Diana Giménez and Julien Prudent and Germain Gillet and Jesús Salgado and Jean-Luc Coll and Abdel Aouacheria},
      title = {Bax-derived membrane-active peptides act as potent and direct inducers of apoptosis in cancer cells},
      journal = {J. Cell Sci.},
      year = {2011},
      volume = {124},
      number = {},
      pages = {556-564},
      doi = {http://dx.doi.org/10.1242/jcs.076745}
    }
    
    Fuertes, G. Baxα5 at lipid membranes: Structure, assembly and pore formation. 2011 PhD Thesis,
    University of Valencia, Paterna, Spain. 
    PhD Thesis URL 
    Abstract: Proteins of the Bcl-2 family control the release of cytochrome c, and other apoptogenic factors, from the mitochondria to the cytosol. Proapoptotic members of the family, like Bax, facilitate such release most likely through the formation of proteolipidic pores in the outer mitochondria membrane (MOM). On the other hand, antiapoptotic members, like Bcl-xL, inhibit the permeabilization of the MOM by blocking Bax activation. The structure of membrane-bound species of Bax responsible for the formation of pores as well as the structure and kinetics of Bax-induced pores remain largely unexplored. In order to gain insight into the pore-forming mechanism of Bax, we have characterized the structure, assembly and pore formation properties of a minimal active domain derived from the fifth helix of Bax, Baxα5. We have found that Baxα5 binds readily to lipid interfaces where it folds mainly as an α-helix. Both FRET and SDS-PAGE data suggest that Baxα5 is able to self-associate in lipid membranes and lipid-mimetic media, respectively. A detailed analysis of the residues involved in Baxα5-Baxα5 interactions suggests the presence of a glycophorin-like dimerization motif supplemented with a tyrosine residue. Interestingly, the number of Baxα5 molecules in the oligomers is dependent on the lipid composition, with mitochondrial lipids inducing higher-order oligomeric assemblies. Baxα5 is also able to interact with Bcl-xL-derived polypeptides. However, such heterotypic associations do not inhibit but rather enhance Baxα5-induced leakage from lipid vesicles. The structure of complexes of Baxα5 with membranes has been extensively studied by infrared spectroscopy and X-ray scattering. Two strategies have been used to best interpret the experimental data in terms of the orientation of Baxα5 with respect to the membrane normal: an implicit orientational landscape model including distributions of the orientation angles and a rigid body modeling approach. Best fits are achieved using a dimeric starting structure which shows an almost flat alignment of Baxα5 in POPC membranes and a more tilted state in DMPC bilayers. Importantly, the orientation displayed by Baxα5 depends on lipid properties, such as fluidity and hydrophobic thickness. In turn, Baxα5 seems to affect the properties of the surrounding lipid matrix. The pore-forming activity of Baxα5 has been addressed both at the ensemble and single-vesicle level. Experiments with suspensions of liposomes allow recording the kinetics of dye release. However, these experiments are restricted to short times. In order to capture the activity of pores at any desired time after peptide/vesicle mixing we have developed two approaches. As a first approach, the quenching of fluorescent lipids by externally added reagents in ensembles of liposomes suggests the presence of pores or membrane defects at long times. Direct visualization of leakage events can be done observing single giant unilamellar vesicles (GUVs) under the fluorescence microscope. Successive leakage events in single GUVs indicate that the vesicle pore-state is maintained over time. Interestingly, individual long-term equilibrium pores have a smaller radius (~2.3 nm) than short-term/kinetic pores suggesting that pores shrink with equilibration. Finally we have shown that the leakage activity of Baxα5 depends on the presence and location of lysine residues within the primary structure.
    BibTeX:
    @phdthesis{Fuertes2011c,
      address = {Spain},
      type = {{PhD}},
      author = {Gustavo Fuertes},
      title = {Baxα5 at lipid membranes: Structure, assembly and pore formation},
      School = {University of Valencia},
      language = {English}
      year = {2011},
      url = {http://www.tdx.cat/bitstream/handle/10803/80916/fuertes.pdf}
    }
    
    Fuertes, G., Pedrueza, E., Abderrafi, K., Abargues, R., Sánchez-Muñoz, O.L., Martínez-Pastor, J., Salgado, J., Jiménez, E. Photoswitchable bactericidal effects from novel silica-coated silver nanoparticles 2011 Proc. SPIE  
    Vol. 8092, V Conference on Medical Laser Applications and Laser-Tissue Interactions, May 24-26, 2011, Munich, Germany, p. 80921M 
    Conference DOI 
    Abstract:

    The enhancement of the electromagnetic field in the surroundings of nanoparticles via surface plasmon resonance offers promising possibilities for biomedical applications. Here we report on the selective triggering of antibacterial activity using a new type of silver nanoparticles coated with silica, Ag@silica, irradiated at their surface plasmon frequency. The nanoparticles are able to bind readily to the surface of bacterial cells, although this does not affect bacterial growing since the silica shell largely attenuates the intrinsic toxicity of silver. However, upon simultaneous exposure to light corresponding to the absorption band of the nanoparticles, bacterial death is triggered selectively on the irradiated zone. Because of the low power density used in the treatments, we discard thermal effects as the cause of cell killing. Instead, we propose that the switched toxicity is due to the enhanced electromagnetic field in the proximity of the nanoparticles, which either directly (through membrane perturbation) or indirectly (through induced photochemical reactions) is able to cause cell death.

    BibTeX:
    @conference{Fuertes2011b,
      author = {Fuertes, Gustavo, Pedrueza, Esteban, Abderrafi, Kamal, Abargues, Rafael, Sánchez-Muñoz, Orlando L., Martínez-Pastor, Juan, Salgado, Jesús, Jiménez, Ernesto},
      title = {Photoswitchable bactericidal effects from novel silica-coated silver nanoparticles},
      journal = {Proc. SPIE},
      year = {2011},
      volume = {8092},
      number = {},
      pages = {80921M},
      url = {http://dx.doi.org/10.1117/12.889675},
      doi = {http://dx.doi.org/10.1117/12.889675}
    }
    
    Fuertes, G., Giménez, D., Esteban-Martín, S., Sánchez-Muñoz, O.L. & Salgado, J. A lipocentric view of peptide-induced pores 2011 Eur. Biophys. J.  
    Vol. 40 (4), pp. 399-415 
    Article DOI
    URL
    Citations 
    Abstract:

    Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, non mediated leakage is not completely improbable. A high activation energy keeps unassisted bilayer permeation normally at a very low frequency, but lipids are able to self organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions like phase coexistence, external stress or perturbation associated to binding of non lipidic molecules. Here we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation of peptides, lipidic pores induced by different types of stress, and spontaneous transient bilayer defects, driven by thermal fluctuations. Within such a lipo-centric framework, amphipathic peptides are best described as pore inducing, rather than pore forming elements. Active peptides bound to membranes can be understood as a source of internal surface tension which facilitates pore formation by diminishing the high activation energy barrier. This first or immediate action of the peptide has some resemblance with a catalysis. However, the presence of membrane active peptides has the additional effect of displacing the equilibrium towards the pore open state, which is then maintained over long times, and reducing the size of initial individual pores. Thus, pore-inducing peptides, regardless of their sequence and oligomeric organization, can be assigned a double role, consisting on increasing the probability of pore formation in membranes to high levels and stabilizing these pores after they appear.

    BibTeX:
    @article{Fuertes2011a,
      author = {Fuertes, Gustavo, Giménez, Diana, Esteban-Martín, Santi, Sánchez-Muñoz, Orlando L., & Salgado, Jesús},
      title = {A lipocentric view of peptide-induced pores},
      journal = {Eur. Biophys. J.},
      year = {2011},
      volume = {40},
      number = {4},
      pages = {399--415},
      doi = {http://dx.doi.org/10.1007/s00249-011-0693-4},
      url = {http://www.springerlink.com/content/07653423428w71m1/}
    }
    
    Fuertes, G., Sánchez-Muñoz, O.L., Pedrueza, E., Abderrafi, K., Salgado, J., Jiménez, E. Switchable Bactericidal Effects from Novel Silica-Coated Silver Nanoparticles Mediated by Light Irradiation 2011 Langmuir  
    Vol. 27 (6), pp. 2826-2833 
    Article DOI
    Supplement
    Citations 
    Abstract:

    Here we report on the triggering of antibacterial activity by a new type of silver nanoparticles coated with porous silica, Ag@silica, irradiated at their surface plasmon resonant frequency. The nanoparticles are able to bind readily to the surface of bacterial cells, although this does not affect bacterial growth since the silica shell largely attenuates the intrinsic toxicity of silver. However, upon simultaneous exposure to light corresponding to the absorption band of the nanoparticles, bacterial death is enhanced selectively on the irradiated zone. Because of the low power density used for the treatments, we discard thermal effects as the cause of cell killing. Instead, we propose that the increase in toxicity is due to the enhanced electromagnetic field in the proximity of the nanoparticles, which indirectly, most likely through induced photochemical reactions, is able to cause cell death.

    BibTeX:
    @article{Fuertes2011,
      author = {Fuertes, Gustavo, Sánchez-Muñoz, Orlando L., Pedrueza, Esteban, Abderrafi, Kamal, Salgado, Jesús, Jiménez, Ernesto},
      title = {Switchable Bactericidal Effects from Novel Silica-Coated Silver Nanoparticles Mediated by Light Irradiation},
      journal = {Langmuir},
      year = {2011},
      volume = {27},
      number = {6},
      pages = {2826-2833},
      url = {http://pubs.acs.org/doi/full/10.1021/la1045282},
      doi = {http://dx.doi.org/10.1021/la1045282}
    }
    
    Guillemin, Y., Lopez, J., Giménez, D., Fuertes, G., García Valero, J., Blum, L., Gonzalo, P., Salgado, J., Girard-Egrot, A. & Aouacheria, A. Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence 2010 PLoS ONE  
    Vol. 5 (2), pp. e9066 
    Article DOI
    Citations 
    Abstract: Background

    The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction.

    Methodology/Principal Findings

    Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death.

    Conclusion/Significance

    BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains.

    BibTeX:
    @article{Guillem2010,
      author = {Guillemin, Yannis AND Lopez, Jonathan AND Giménez, Diana AND Fuertes, Gustavo AND Valero, Juan García AND Blum, Loïc AND Gonzalo, Philippe AND Salgado, Jesús AND Girard-Egrot, Agnès AND Aouacheria, Abdel},
      title = {Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence},
      journal = {PLoS ONE},
      year = {2010},
      volume = {5},
      number = {2},
      pages = {e9066},
      doi = {http://dx.doi.org/10.1371/journal.pone.0009066}
    }
    
    Esteban-Martín, S., Strandberg, E., Salgado, J. & Ulrich, A. S. Solid state NMR analysis of peptides in membranes: influence of dynamics and labeling scheme 2010 BBA-Biomembranes
    Vol. 1798 (2), pp. 252-257 
    Article DOI
    Citations 
    Abstract: The functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These “static” structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that the reliability of a simplified static or an advanced dynamic data analysis depends critically on the choice of isotope labeling scheme used. Two distinctly different scenarios have to be considered. When the labels are placed on the side chains of a helical peptide (such as a CD3- or CF3- group attached to the Cα-Cβ bond), their nuclear spin interaction tensors are very sensitive to motional averaging. If this effect is not properly accounted for, the helix tilt angle tends to be severely underestimated. At the same time, the analysis of labels in the side chains allows to extract valuable dynamical information about whole-body fluctuations of the peptide helix in the membrane. On the other hand, the alternative labeling scheme where 15N-labels are accommodated within the peptide backbone, will yield nearly correct helix tilt angles, irrespective as to whether dynamics are taken into account or not.
    BibTeX:
    @article{Esteban2010,
      author = {Santi Esteban-Martín and Erik Strandberg and Jesús Salgado and Anne S. Ulrich},
      title = {Solid state NMR analysis of peptides in membranes: influence of dynamics and labeling scheme},
      journal = {Biochim. Biophys. Acta},
      year = {2010},
      volume = {1798},
      number = {2},
      pages = {252-257},
      url = {10.1016/j.bpj.2009.02.040}
      doi = {10.1016/j.bpj.2009.02.040}
    }
    
    Fuertes, G., García-Síez, A.J., Esteban-Martín, S., Giménez, D., Sánchez-Muñoz, O.L., Schwille, P. & Salgado, J. Pores formed by Baxα5 relax to a smaller size and keep at equilibrium 2010 Biophys. J.  
    Vol. 99 (9), pp. 2917-2925 
    Article DOI
    Supplement
    Citations 
    Abstract:

    Pores made by amphipathic cationic peptides, like antimicrobials and fragments of pore-forming proteins, are typically studied by either kinetics of vesicle leakage after peptide addition or structural measurements in reconstituted peptide-lipid systems. In the first case the pores have been considered transient phenomena that allow the relaxation of the peptide-membrane system. In the second, they correspond to equilibrium structures at minimum free energy. Here we reconcile both approaches by investigating the pore activity of the α5 fragment from the proapoptotic protein Bax (Baxα5) before and after equilibrium of peptide/vesicle complexes. Quenching assays on suspensions of large unilamelar vesicles (LUVs), suggest that in presence of Baxα5 the vesicles maintain a leaky state for hours and under equilibrium conditions. Stable pores were proved and analyzed in detail on single giant unilamelar vesicles (GUVs) by monitoring the entrance of dyes added at different times after incubation with the peptide. When GUVs get in contact with Baxα5, leakage starts stochastically, delayed by some variable time, and in the majority of cases proceeds rapidly to completion. After hours in presence of the peptide the same individual GUVs refilling completely at first-instance maintain a porated state, which can be observed in subsequent leak-in events for serially added dyes. However, such long-term pores were of smaller size than the initial equilibration pores. Stable pores were also detected in GUVs made in presence of Baxα5. These latter pores can be considered equilibrium states and may correspond to the structures measured previously in bilayer stacks. Although pore formation may occur as a kinetic process, equilibrium pores can be functionally relevant structures, especially in highly regulated systems, like the apoptotic mitochondrial pores induced by Bax.

    BibTeX:
    @article{Fuertes2010b,
      author = {Fuertes, Gustavo, García-Sáez, Ana J., Esteban-Martín, Santi, Giménez, Diana,  Sánchez-Muñoz, Orlando L., Schwille, Petra & Salgado, Jesús},
      title = {Pores formed by Baxa5 relax to a smaller size and keep at equilibrium},
      journal = {Biophys. J.},
      year = {2010},
      volume = {99},
      number = {9},
      pages = {2917-2925},
      doi = {http://dx.doi.org/10.1016/j.bpj.2010.08.068}
    }
    
    Fuertes, G., Manor, J., Esteban-Martín, S., Arkin, I. T. & Salgado, J. Structure Of Complexes Of Helix-5 From Bax With Lipid Membranes 2009 Biophys. J.
    Vol. 96 (3, S1), Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA, p. 159a 
    Conference DOI 
    Abstract: Bax is a proapoptotic protein implicated in the release of cell-death activating factors from the mitochondrial intermembrane space. Although the structure of the membrane-bound forms of Bax is unknown, it has been proposed to form proteolipidic pores. Studies with synthetic lipid vesicles have shown that fragments encompassing helix-5 of Bax retain a membrane permeabilization ability that is similar to that of the full-length protein. Here we report on the structure of peptide-membrane complexes formed by a Bax helix-5 peptide and lipid bilayers. The relative orientation of the peptide and the lipids are determined using site-specific infrared spectroscopy, assisted by isotopic labeling of backbone groups with the 13C=18O probe. The peptide is highly α-helical in all lipid membranes studied, and its orientation reveals different binding modes that depend on the bilayer phase state. In partially fluid POPC bilayers helix-5 of Bax lies almost parallel with respect to the membrane plane, most likely interacting at the level of the interface. However, in gel phase DMPC bilayers the peptide adopts a tilted orientation, which suggests a deeper insertion in the membrane. In turn infrared spectroscopy and X-ray diffraction data show that in some instances the Bax helix-5 peptide influences the phase transition properties of the lipids by increasing membrane fluidity. Taken together, these effects can be related with the membrane perturbation properties of the Bax helix-5 fragment and with its mechanism of action as a molecule inducing the formation of lipidic pores.
    BibTeX:
    @conference{Fuertes2010a,
      author = {Gustavo Fuertes and Joshua Manor and Santi Esteban-Martín and Isaiah T. Arkin and Jesús Salgado},
      title = {Structure Of Complexes Of Helix-5 From Bax With Lipid Membranes},
      booktitle = {Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA},
      journal = {Biophys. J.},
      year = {2009},
      volume = {96},
      number = {3, S1},
      pages = {159a},
      url = {http://www.sciencedirect.com/science/article/B94RW-4VK7BB2-11C/2/5f3c30483f97873d0bc0653bcd71bdbe},
      doi = {http://dx.doi.org/10.1016/j.bpj.2008.12.726}
    }
    
    Fuertes, G., Giménez, D., Esteban-Martín, S., García-Sáez, A. J., Sánchez, O. & Salgado, J. Role of Membrane Lipids for the Activity of Pore forming Peptides and Proteins 2010 In Proteins: Membrane Binding and Pore Formation (series: Advances in Experimental Medicine and Biology, vol. 677), Anderluh, G. & Lakey, J. (eds.). Springer & Landes Bioscience, New York, pp 31-55. Chapter DOI
    URL
    Citations 
    Abstract: Bilayer lipids, far from being passive elements, exert multiple roles in polypeptide dependent pore formation. Lipids participate at all stages of the formation of pores, by providing the binding site for proteins and peptides, conditioning their active structure and modulating the molecular reorganization of the membrane complex. Such general functions of lipids superimpose to other particular roles, from electrostatic and curvature effects to more specific actions in cases like cholesterol, sphingolipids or cardiolipin. Pores are natural phenomena in lipid membranes. Driven by membrane fluctuations and packing defects, transient water pores are related to spontaneous lipid flip-flop and non-assisted ion permeation. In the absence of proteins or peptides, these are rare short living events, with properties dependent on the lipid composition of the membrane. Their frequency increases under conditions of internal membrane disturbance of the lipid packing, like in the presence of membrane-bound proteins or peptides. These latter molecules, in fact, form dynamic supramolecular assemblies together with the lipids, and transmembrane pores are one of the possible structures of the complex. Active peptides and proteins can thus be considered inducers or enhancers of pores which increase their probability and lifetime by modifying the thermodynamic membrane balance. This includes destabilizing the membrane lamellar structure, lowering the activation energy for pore formation and stabilizing the open pore structure.
    BibTeX:
    @incollection{Fuertes2010,
    	author = {Gustavo Fuertes and Diana Gimenez and Santi Esteban-Martin and Ana J Garca-Saez and Orlando Sanchez and Jesus Salgado},
    	title = {Role of Membrane Lipids for the Activity of Pore forming Peptides and Proteins},
    	booktitle = {Proteins: Membrane Binding and Pore Formation},
    	series = {Advances in Experimental Medicine and Biology},
    	volume = {677},
    	editor = {Gregor Anderluh and Jeremy Lakey},
    	publisher = {Springer and Landes Bioscience},
    	address = {New York},
    	year = {2010},
    	pages = {31--55},
    	doi = {http://dx.doi.org/10.1007/978-1-4419-6327-7_4},
    	url = {http://www.springerlink.com/content/j887215w411t1195/}
    }
    
    García-Sáez, A. J., Fuertes, G., Suckale, J. & Salgado, J. Permeabilization of the Outer Mitochondrial Membrane by Bcl‑2 Proteins 2010 In Proteins: Membrane Binding and Pore Formation (series: Advances in Experimental Medicine and Biology, vol. 677), Anderluh, G. & Lakey, J. (eds.). Springer & Landes Bioscience, New York, pp 91-105. Chapter DOI
    URL
    Citations 
    Abstract: The proteins of the Bcl‑2 family regulate the release of the apoptotic factors from mitochondria during apoptosis, a key event in physiological cell death. Although their molecular mechanisms remain unclear, the Bcl‑2 proteins have been proposed to directly control the permeability of the outer mitochondrial membrane by pore formation. Indeed, they share structural features with the pore forming domains of some bacterial toxins and they can give rise to proteolipidic pores in model membranes. The complex level of regulation needed to decide the fate of the cell is achieved by an intricate interaction network between different members of the family. Current models consider multiple parallel equilibria of activation and inhibition that determine whether the permeabilization of the mitochondrial outer membrane is induced or not.
    BibTeX:
    @Chapter{Garcia2010,
    	author = {Ana J. Garcia-Saez and Gustavo Fuertes and Jacob Suckale and Jesus Salgado},
    	title = {Permeabilization of the Outer Mitochondrial Membrane by Bcl‑2 Proteins},
    	booktitle = {Proteins: Membrane Binding and Pore Formation},
    	series = {Advances in Experimental Medicine and Biology},
    	volume = {677},
    	editor = {Gregor Anderluh and Jeremy Lakey},
    	publisher = {Springer and Landes Bioscience},
    	address = {New York},
    	year = {2010},
    	pages = {91--105},
    	doi = {http://dx.doi.org/10.1007/978-1-4419-6327-7_8},
    	url = {http://www.springerlink.com/content/t431222450641018/}
    }
    
    Strandberg, E., Esteban-Martín, S., Salgado, J. & Ulrich, A. S. Orientation and dynamics of peptides in membranes calculated from 2H-NMR Data 2009 Biophys. J.
    Vol. 96(8), pp. 3223-3232 
    Article DOI
    Citations 
    Abstract: Solid state 2H-NMR is routinely used to determine the alignment of membrane-bound peptides. Here we demonstrate that it can also provide a quantitative measure of the fluctuations around the distinct molecular axes. Using several dynamical models with increasing complexity, we re-analyzed published 2H-NMR data on two representative alpha-helical peptides: (i) The amphiphilic antimicrobial peptide PGLa which permeabilizes membranes by going from a monomeric surface-bound to a dimeric tilted state, and finally inserting as an oligomeric pore. (ii) The hydrophobic WALP23 is a typical transmembrane segment, but previous analysis had yielded helix tilt angles much smaller than expected from hydrophobic mismatch and MD simulations. Their 2H-NMR data were deconvoluted in terms of the two main helix orientation angles (representing the time-averaged peptide tilt and azimuthal rotation), as well as the amplitudes of fluctuation about the corresponding molecular axes (providing the dynamic picture). The mobility of PGLa is found to be moderate, and to correlate well with the respective oligomeric states. WALP23 fluctuates more vigorously, now in full agreement with the MD simulations and mismatch predictions. The analysis demonstrates that when fitting 2H-NMR data to extract peptide orientation angles an explicit representation of the peptide rigid-body angular fluctuations should be included.
    BibTeX:
    @article{Strandb2009,
      author = {Erik Strandberg and Santi Esteban-Martín and Jesús Salgado and Anne S. Ulrich},
      title = {Orientation and Dynamics of Peptides in Membranes Calculated from 2H-NMR Data},
      journal = {Biophys. J.},
      year = {2009},
      volume = {96},
      number = {8},
      pages = {3223-3232},
      doi = {http://dx.doi.org/10.1016/j.bpj.2009.02.040}
    }
    
    Strandberg, E., Esteban-Martín, S., Salgado, J. & Ulrich, A. S. Influence of Dynamics on The Analysis of Solid-State NMR Data From Membrane-bound Peptides 2009 Biophys. J.
    Vol. 96 (3, S1), Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA, pp. 408a-409a  
    Conference DOI 
    Abstract: By isotope labeling of membrane-bound peptides, typically with 2H, 19F, or 15N, solid-state NMR experiments can yield data from which the orientation of peptides in a native membrane environment can be determined. Such an orientation is defined by a tilt angle and an azimuthal rotation angle. Here we show that to obtain correct values of the orientation angles, it is important to include dynamics in the analysis of the NMR data. Nevertheless the effects of dynamics are different depending on the type of isotope labeling and NMR experiment considered.

    To analyze the influence of dynamics in detail, we generated virtual NMR observables using a model peptide undergoing explicit Gaussian fluctuations of the orientation angles. For simulated 2H- or 19F-NMR data, even moderate motions were found to have a large influence, as calculated tilt values are consistently much too small, unless dynamics is properly considered. A simple dynamic model, including a molecular order parameter scaling factor, gives good results only for moderately mobile peptides, while for high mobility cases the correct tilt is only obtained by re-introducing the explicit Gaussian fluctuations in the fitting functions. In contrast, 15N-NMR data appear to be less sensitive to rigid-body peptide motions, and PISEMA spectra can give correct orientations even for highly mobile peptides, and assuming a static model for the analysis. The differences are due to the different orientation of the tensors of 2H- and 19F-labels, placed on peptide side chains, compared to the orientation of the 15N tensor, placed on amide backbone groups.

    We conclude that dynamics should be included in the analysis of solid-state NMR data of membrane-bound peptides. Not only does this give more accurate orientations, but it can also provide information about the dynamics of the peptide.

    BibTeX:
    @conference{Strandb2009a,
      author = {Erik Strandberg and Santi Esteban-Martin and Jesus Salgado and Anne S. Ulrich},
      title = {Influence of Dynamics on The Analysis of Solid-State NMR Data From Membrane-bound Peptides},
      booktitle = {Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA},
      journal = {Biophys. J.},
      year = {2009},
      volume = {96},
      number = {3, S1},
      pages = {408a-409a},
      url = {http://www.sciencedirect.com/science/article/B94RW-4VK7BB2-2N4/2/811824ee4bcab81ff3bcab1498c2b0fd},
      doi = {http://dx.doi.org/10.1016/j.bpj.2008.12.2083}
    }
    
    Salgado, J. From Hydrophobic Matching to Interfacial Tuning: New Ideas for the Mutual Adaptation Between Membranes and Peptides 2009 Biophys. J.
    Vol. 96 (3, S1), Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA, p. 2a 
    Conference DOI 
    Abstract: It is widely accepted that membrane proteins and lipid bilayers are complementary in terms of the distribution in space of their hydrophobic and polar regions. Similarly, it is also accepted that the hydrophobic parts of the protein and the membrane must adapt to each other. Classically these ideas are rationalized under the concept of hydrophobic matching, which predicts a number of possible mechanisms by which proteins can vary their effective hydrophobic length, or membranes can change their hydrophobic thickness. Such effects have been studied in detail for simplified systems, like transmembrane peptides or protein fragments, which generally show that optimizing peptide orientation is the principal adaptation response.

    Based on simple computational methods, we show that the relative positioning, including orientation, of a peptide in a membrane can be easily and accurately predicted if the bilayer interfaces are taken into account. This allows studying in detail the adaptations of peptides to membranes, showing that, together with the classical coarse adjustment achieved by changes of the peptide tilt, there can be fine tuned adjustments through the azimuthal rotation. The latter tuning effect occurs mainly by optimizing positions of residues near the interface, and because it involves small changes of free energy, it provides a mechanism for high peptide dynamics. Additionally it strengthens the importance of the bilayer interface for the mutual adaptation of membranes and proteins and gives a new framework for the definition of so called flanking (or anchoring) residues.

    BibTeX:
    @conference{Salgado2009,
      author = {Jesús Salgado},
      title = {From Hydrophobic Matching to Interfacial Tuning: New Ideas for the Mutual Adaptation Between Membranes and Peptides},
      booktitle = {Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA},
      journal = {Biophys. J.},
      year = {2009},
      volume = {96},
      number = {3, S1},
      pages = {2a},
      url = {http://www.sciencedirect.com/science/article/B94RW-4VK7BB2-8/2/ae70e37f21dcb355340c428a397d8f16},
      doi = {http://dx.doi.org/10.1016/j.bpj.2008.12.013}
    }
    
    Esteban-Martín, S. Unravelling the orientation and dynamics of membrane peptides. 2009 PhD Thesis,
    University of Valencia, Paterna, Spain. 
    PhD Thesis URL 
    Abstract: During the last 10 years a number of spectroscopic techniques have developed and matured enough to allow the precise determination of the orientation of membrane peptides based on experimental measures. Because most of the available experimental orientational information is based on static models, this Thesis aims to expand this view and focus on the dynamics of the peptide-membrane systems. Of special interest are the model transmembrane peptides from the WALP/WLP and KALP/KLP series, designed to understand membrane peptide interactions in detail. Despite their apparent simplicity, these systems have become controversial as their tilt in the membrane, determined from 2H NMR data, has been reported to be smaller than expected and barely reacting to mismatch, which contrast to that observed for a number of natural peptides. This Thesis is organized as follows: Chapter 7 addresses the origin of discrepancies encountered between computational methods and 2H-NMR spectroscopy for the determination of peptide orientation. This is studied by molecular dynamics simulations. Chapters 8 and 9 are a systematic analysis of the effect of whole-body peptide motions on 2H-splittings and 2D PISA wheels. In Chapter 10 the thermodynamically available positional and orientational states of (rigid) peptides in implicit membranes are explored based on the free energy of partitioning aminoacid residues into the interface or the hydrophobic parts of bilayers. In Chapter 11 we investigate the capability of a random distribution of lipids and a peptide to self-organized in water into a lipid bilayer. We show that this method allows obtaining non-biased membrane-protein systems for the study of preferred membrane-peptide binding modes at atomic detail.
    BibTeX:
    @phdthesis{Esteban2009d,
      address = {Spain},
      type = {{PhD}},
      author = {Santi Esteban-Martín},
      title = {Unravelling the orientation and dynamics of membrane peptides},
      School = {University of Valencia},
      language = {English}
      year = {2009},
      url = {https://www.educacion.gob.es/teseo/mostrarRef.do?ref=589638}
    }
    
    Esteban-Martín, S., Strandberg, E., Fuertes, G., Ulrich, A. S. & Salgado, J. Experiments Meet Hydrophobic Mismatch: A Re-evaluation Of The Orientation Of Model Transmembrane Peptides From Solid-State NMR 2009 Biophys. J.
    Vol. 96 (3, S1), Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA, p. 159a 
    Conference DOI 
    Abstract: The basic physical rules underlying the organization of biological membranes can be gathered under the simple, but powerful, concept of hydrophobic mismatch. For example, the mutual adjustment of the lipid and protein hydrophobic lengths can be related with the existence of lipid rafts and explain discrete secretory pathways in the Golgi apparatus. The orientation of membrane protein fragments is predicted to follow the same hydrophobic mismatch principles, as illustrated by some experiments and molecular dynamics simulations. However, this appears to be challenged by results of solid-state 2H NMR experiments on model transmembrane peptides, displaying tilt angle values unexpectedly small and weakly reacting to changes of the lipid bilayer thickness.

    Here we bridge theory and experiments to show that previous 2H NMR experimental data of model transmembrane peptides in membranes of different thickness can be re-interpreted by using alternative models which consider explicit rigid-body peptide fluctuations. The result is a new set of tilts which follows nicely the hydrophobic mismatch expectations, and is coherent with molecular dynamics simulations as well as with other mismatch studies conducted with natural protein fragments.

    BibTeX:
    @conference{Esteban2009c,
      author = {Santi Esteban-Martin and Erik Strandberg and Gustavo Fuertes and Anne S. Ulrich and Jesus Salgado},
      title = {Experiments Meet Hydrophobic Mismatch: A Re-evaluation Of The Orientation Of Model Transmembrane Peptides From Solid-State NMR},
      booktitle = {Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA},
      journal = {Biophys. J.},
      year = {2009},
      volume = {96},
      number = {3, S1},
      pages = {159a},
      url = {http://www.sciencedirect.com/science/article/B94RW-4VK7BB2-119/2/8da8104be3ac433c9a5250ccc1e60317},
      doi = {http://dx.doi.org/10.1016/j.bpj.2008.12.724}
    }
    
    Esteban-Martín, S., Giménez, D. Fuertes, G. & Salgado, J. Orientational landscapes of peptides in membranes: Prediction of 2H NMR couplings in a dynamic context 2009 Biochemistry
    Vol. 48 (48), pp. 11441–11448 
    Article DOI
    Citations 
    Abstract: Unlike soluble proteins, membrane polypeptides face an anisotropic milieu. This imposes restrains to their orientation and provides a reference that makes structure prediction tractable by minimalistic thermodynamic models. Here we use this framework to build orientational distributions of monomeric membrane-bound peptides and to predict their expected solid-state 2H NMR quadrupolar couplings when labeled at specific side chain positions. Using a complete rigid-body sampling of configurations relative to an implicit lipid membrane, peptide free energy landscapes are calculated. This allows obtaining probability distributions of the peptide tilt, azimuthal rotation and depth of membrane insertion. The orientational distributions are broad and originate from an interplay between the three relevant rigid-body degrees of freedom, which allows populating multiple states in shallow free energy minima. Remarkably, only when the orientational distributions are taken into account, we obtain a close correlation between predicted 2H NMR splittings and values measured in experiments. Such a good correlation is not seen with splittings calculated from single configurations, being either the averaged or the lowest free energy state, showing that are distributions, rather than single structures, which best define the peptide-membrane systems. Moreover, we propose that these distributions contribute to the understanding of the rigid-body dynamics of the system.
    BibTeX:
    @article{Esteban2009b,
      author = {Santi Esteban-Martín and Diana Giménez and Gustavo Fuertes and Jesús Salgado},
      title = {Orientational landscapes of peptides in membranes: Prediction of 2H NMR couplings in a dynamic context},
      journal = {Biochemistry},
      year = {2009},
      volume = {48},
      number = {48},
      pages = {11441–11448},
      doi = {http://dx.doi.org/10.1021/bi901017y}
    }
    
    Esteban-Martín, S., Risselada, H. J., Salgado, J. & Marrink, S. J. Stability of Asymmetric Lipid Bilayers Assessed by Molecular Dynamics Simulations 2009 J. Am. Chem. Soc.
    Vol. 131 (42), pp. 15194–15202 
    Article DOI
    Citations 
    Abstract: The asymmetric insertion of amphiphiles into biological membranes compromises the balance between the inner and outer monolayers. As a result, area expansion of the receiving leaflet and curvature strain may lead to membrane permeation, shape changes, or membrane fusion events. We have conducted both atomistic and coarse-grained molecular dynamics simulations of dipalmitoyl-phosphatidylcholine (DPPC) bilayers to study the effect of an asymmetric distribution of lipids between the two monolayers on membrane stability. Highly asymmetric lipid bilayers were found to be surprisingly stable within the submicrosecond time span of the simulations. Even the limiting case of a monolayer immersed in water ruptured spontaneously only after at least 20 ns simulation. A thermal shock could destabilize these kinetically trapped states. We also studied mixed systems composed of DPPC and short tail diC8PC lipids, showing that the presence of the cone-shaped short tail lipid facilitates the release of tension in the asymmetric systems via formation of a transmembrane pore. Thus, asymmetric area expansion and curvature stress cooperate to yield bilayer disruption. It appears that, although asymmetric area expansion destabilizes the bilayer structure, the activation energy for transmonolayer lipid re-equilibration is increased. Such a large kinetic barrier can be reduced by lipids with positive spontaneous curvature. These effects are important at the onset of bilayer destabilization phenomena, such as lipid pore formation and membrane fusion, and should be considered for the mechanism of induction of such processes by peptides and proteins.
    BibTeX:
    @article{Esteban2009a,
      author = {Santi Esteban-Martín and H. Jelger Risselada and Jesús Salgado and Siewert J. Marrink},
      title = {Stability of Asymmetric Lipid Bilayers Assessed by Molecular Dynamics Simulations},
      journal = {J. Am. Chem. Soc.},
      year = {2009},
      volume = {131},
      number = {42},
      pages = {15194–15202},
      doi = {http://dx.doi.org/10.1021/ja904450t}
    }
    
    Esteban-Martín, S., Strandberg, E., Fuertes, G., Ulrich, A. S. & Salgado, J. Influence of whole-body dynamics on 15N PISEMA NMR spectra of membrane peptides: a theoretical analysis. 2009 Biophys. J.
    Vol. 96(8), pp. 3233-3241 
    Article DOI
    Citations 
    Abstract: Membrane proteins and peptides exhibit a preferred orientation in the lipid bilayer while fluctuating in an anisotropic manner. Both the orientation and the dynamics have direct functional implications, but motions are usually not accessible and structural descriptions are generally static. Using simulated data, we analyse systematically the impact of whole-body motions on the peptide orientations calculated from 2D PISEMA NMR. Fluctuations are found to have a significant effect on the observed spectra. Nevertheless, wheel-like patterns are still preserved, and it is possible to determine the average peptide tilt and azimuthal rotation angles using simple static models for the spectral fitting. For helical peptides undergoing large amplitude fluctuations, as in the case of transmembrane monomers, improved fits can be achieved by using an explicit dynamics model which includes Gaussian distributions of the orientational parameters. This method allows extracting the amplitudes of fluctuations of the tilt and azimuthal rotation angles. The analysis is further demonstrated by generating first a virtual PISEMA spectrum from a molecular dynamics trajectory of the model peptide WLP23 in a lipid membrane. That way, the dynamics of the system from which the input spectrum originates is completely known at atomic detail, and can thus be directly compared with the dynamical output obtained from the fit. We find that fitting our dynamics model to the PISA wheel gives an accurate description of the amplitude of underlying motions, together with the average peptide orientation.
    BibTeX:
    @article{Esteban2009,
      author = {Santi Esteban-Martín and Erik Strandberg and Gustavo Fuertes and Anne S. Ulrich and Jesús Salgado},
      title = {Influence of whole-body dynamics on 15N PISEMA NMR spectra of membrane peptides: a theoretical analysis.},
      journal = {Biophys. J.},
      year = {2009},
      volume = {96},
      number = {8},
      pages = {3233-3241},
      doi = {http://dx.doi.org/10.1016/j.bpj.2008.12.3950}
    }
    
    Afonin, S., Grage, S. L., Ieronimo, M., Maisch, D., Wadhwani, P., Mykhailiuk, P. K., Salgado, J., Komarov, I. V. & Ulrich, A. S. The Alignment of Membrane-Active Peptides Depends on the Lipid Phase State as Viewed by solid state 19F-NMR 2009 Biophys. J.
    Vol. 96 (3, S1), Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA, p. 156a 
    Conference DOI 
    Abstract: Amphipathic membrane-active peptides (antimicrobial, hemolytic, cell-penetrating, fusogenic, etc.) achieve their functions by distinct interaction with lipid bilayers. Some typical structural modes are described in terms of models like the “barrel stave”, “toroidal pore”, “carpet” etc. These models are related to the alignment states of the peptides in the lipid bilayers (surface bound “S-state”, inserted “I-state” or tilted “T-state”), which can be readily characterized by solid state NMR. When determining such alignment, factors like peptide/lipid ratio, charge of the bilayer surface, thickness of the bilayer core, presence of cholesterol, and humidity are typically investigated. Yet, the lipid phase state as an explicit variable parameter has not received much attention so far. Here, we demonstrate that a change in the lipid phase can directly trigger the re-alignment of many peptides. Several representative examples are illustrated here: PGLa, PGLa/Magainin, gramicidin S, SAP and alamethicin. In macroscopically oriented DMPC bilayers, using highly-sensitive 19F-NMR we have monitored the changes between known alignment states of these peptides as a function of temperature, covering both the gel and liquid-crystalline states of DMPC. We show that for all peptides studied the alignment in the gel-state differs from the one in the liquid-crystalline bilayers and can be reversibly changed by passing through the lipid phase transition temperature. The relevance of these finding for the phase state of native biological membranes and interactions of membrane-active peptides with them will be discussed.
    BibTeX:
    @conference{Afonin2009,
      author = {Sergii Afonin and Stephan L. Grage and Marco Ieronimo and Daniel Maisch and Parvesh Wadhwani and Pavel K. Mykhailiuk and Jesus Salgado and Igor V. Komarov and Anne S. Ulrich},
      title = {The Alignment of Membrane-Active Peptides Depends on the Lipid Phase State as Viewed by solid state 19F-NMR},
      booktitle = {Biophysical Society 53rd Annual Meeting, Feb. 28-Mar. 4 2009, Boston, MA, USA},
      journal = {Biophys. J.},
      year = {2009},
      volume = {96},
      number = {3, S1},
      pages = {156a},
      url = {http://www.sciencedirect.com/science/article/B94RW-4VK7BB2-10V/2/3558e716caa8715546133cb3aa9bc32b},
      doi = {http://dx.doi.org/10.1016/j.bpj.2008.12.710}
    }
    
    Pantoja-Uceda, D., Pastor, M.T., Salgado, J., Pineda-Lucena, A. & Pérez-Payá, E. Design of a bivalent peptide with two independent elements of secondary structure able to fold autonomously. 2008 J. Pept. Sci.
    Vol. 14(7), pp. 845-854 
    Article DOI
    Citations 
    Abstract: This article describes a strategy to develop, starting from a de novo design, bivalent peptides containing two different (alpha-helix and beta-hairpin) and independent secondary-structure elements. The design was based on the use of conformationally restricted peptide libraries. Structural characterization by NMR revealed that the peptides were stable and did not show any long-range NOE interactions between the N-terminal beta-hairpin and the C-terminal alpha-helix. These results suggest that the two elements of secondary structure are stable and well folded. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.
    BibTeX:
    @article{Pantoja2008,
      author = {David Pantoja-Uceda and M. Teresa Pastor and Jesús Salgado and Antonio Pineda-Lucena and Enrique Pérez-Payá},
      title = {Design of a bivalent peptide with two independent elements of secondary structure able to fold autonomously.},
      journal = {J. Pept. Sci.},
      year = {2008},
      volume = {14},
      number = {7},
      pages = {845-854},
      doi = {http://dx.doi.org/10.1002/psc.1015}
    }
    
    Afonin, S., Dürr, U.H., Wadhwani, P., Salgado, J. & Ulrich, A.S. Solid State NMR Structure Analysis of the Antimicrobial Peptide Gramicidin S in Lipid Membranes: Concentration-Dependent Re-alignment and Self-Assembly as a β-Barrel 2008 In Bioactive Conformation II (series: Topics in Current Chemistry, vol. 273), Peters, T. (ed.). Springer International, Berlin/Heidelberg, pp. 139-154. Chapter DOI
    URL
    Citations 
    Abstract: Antimicrobial peptides can kill bacteria by permeabilizing their cell membrane, as these amphiphilic molecules interact favourably with lipid bilayers. This mechanism of action is attributed either to the formation of a peptide “carpet” on the membrane surface, or to a transmembrane pore. However, the structure of such a pore has not yet been resolved under relevant conditions. Gramicidin S is a symmetrical cyclic β-sheet decapeptide, which has been previously shown by solid state NMR to lie flat on the membrane surface at low peptide:lipid ratios (≤ 1:80). Using highly sensitive 19F-NMR, supported by 15N-labelling, we found that gramicidin S can flip into an upright transmembrane alignment at high peptide:lipid ratios (≥ 1:40). Orientational NMR constraints suggest that the peptide may self-assemble as an oligomeric β-barrel pore, which is stabilized by intermolecular hydrogen bonds. Comparison of different model membranes shows that the observed re-alignment is favoured in thin bilayers with short-chain lipids, especially near the chain melting temperature, whereas long-chain lipids suppress pore formation. Based on the oligomeric structural model and the conditions of pore formation, guidelines may now be derived for rationally designing peptide analogues as antibiotics with improved selectivity and reduced side effects.
    BibTeX:
    @Chapter{Afonin2008,
      author = {Sergii Afonin and Ulrich H.N. Dürr and Parvesh Wadhwani and Jesus Salgado and Anne S. Ulrich},
      title = {Solid State NMR Structure Analysis of the Antimicrobial Peptide Gramicidin S in Lipid Membranes: Concentration-Dependent Re-alignment and Self-Assembly as a β-Barrel},
      booktitle = {Bioactive Conformation II},
      series = {Topics in Current Chemistry},
      editor = {Peters, Thomas},
      publisher = {Springer International}
      address = {Berlin/Heidelberg},,
      year = {2008},
      volume = {273},
      url = {http://www.springerlink.com/content/82xl3898111q7660},
      doi = {http://dx.doi.org/10.1007/128_2007_20}
    }
    
    Strandberg, E., Estaban, S., Salgado, J. & Ulrich, A.S. Orientation of Membrane Bound Peptides Studied with Solid State NMR 2008 Biophys. J.
    Vol. 94 (2, S), Biophysical Society 52th Annual Meeting & 16th International Biophysics Congress, February 2-6, 2008, Long Beach, CA, USA, p. 259 
    Conference URL 
    Abstract: The tilt angles of transmembrane model peptides have been subject of debate; especially has MD simulations given larger tilt angles for WALP model peptides than previously published values calculated from 2H-NMR. We show that the values calculated from 2H-NMR data depend on the model used and for large rotational motion around the peptide, tilt angles close to those from MD simulations can be obtained. PISEMA NMR experiments on 15N-labeled WLP peptides are performed to resolve the ambiguity.

    The influence of the dynamics model used on the calculated orientation from 2H-NMR data on 3,3,3-2H-alanine labeled WALP and PGLa peptides is investigated by comparing models of increasing complexity. In the simplest model only a tilt angle (t) and an azimuthal angle (r) are used. Considerably improved fits are obtained when dynamics in included as an order parameter which scales all data with a factor between 0 and 1. To better model peptide mobility, additional parameters were used. ( i) Peptide mobility around the peptide axis was modeled with a Gaussian distribution of r angles, centered at r0 and with width sr. (ii) A corresponding distribution of t angles was introduced. (iii) Simultaneous distributions of both t and r were considered.

    From calculations using these models on 2H-NMR data, it was found that there is an ambiguity of the tilt angle of peptides in a transmembrane orientation when a distribution of azimuthal angles is introduced. This is explained by the form of the helical wave being the same for different sets of parameters. The ambiguity is not seen for peptides in a surface-bound or slightly tilted state, where the orientation is well-defined independent of the dynamics model. A distribution of tilt angles does not give similar effects.

    BibTeX:
    @conference{Strandb2008,
      author = {Erik Strandberg and Santi Estaban-Martín and Jesús Salgado and Anne S. Ulrich},
      title = {Orientation of Membrane Bound Peptides Studied with Solid State NMR},
      booktitle = {Biophysical Society 52th Annual Meeting & 16th International Biophysics Congress, February 2-6, 2008, Long Beach, CA, USA},
      journal = {Biophys. J.},
      year = {2008},
      volume = {94},
      pages = {259},
      url = {http://www.cell.com/biophysj/fulltext/S0006-3495%2808%2979029-5}
    }
    
    Salgado, J. & Esteban-Martín, S. A Dynamic View Of Hydrophobic Matching From A Free Energy-based Positional And Orientational Landscape Of Peptides In Membranes 2008 Biophys. J.
    Vol. 94 (2, S), Biophysical Society 52th Annual Meeting & 16th International Biophysics Congress, February 2-6, 2008, Long Beach, CA, USA, p. 659 
    Conference URL 
    Abstract: The anisotropic environment of the lipid bilayer constraints the molecular position and orientation, allowing the use of minimalistic thermodynamic models for the structural characterization of peptide/membrane complexes. Based on the Wimley-White double scale of partition free energies, a landscape of positional and orientational states for monomeric peptides in membranes is be calculated, from which we select stability-based probability distributions. For the orientational angles (helix tilt and self-rotation) the distributions are broad and asymmetric, in excellent agreement with available molecular dynamics simulations and with important implications for the correct interpretation of experiments. Such distributions allow a close prediction of the experimental observables for a number of model and natural membrane peptides, whether amphipathic and bond parallel at the membrane interface or inserted across the membrane in a tilted configuration. Moreover, we show that the predicted distributions result from optimizing the position of peptide residues with respect to the hydrophobic, the interface and the bulk-water regions through a complex interplay of displacement along the membrane normal, tilt and rotation. This leads to a re-definition of the hydrophobic matching peptide adaptation, stressing its dynamic character and so far unconsidered roles of the membrane interface and peptide rotation.
    BibTeX:
    @conference{Salgado2008,
      author = {Jesús Salgado and Santi Esteban-Martín},
      title = {A Dynamic View Of Hydrophobic Matching From A Free Energy-based Positional And Orientational Landscape Of Peptides In Membranes},
      booktitle = {Biophysical Society 52th Annual Meeting & 16th International Biophysics Congress, February 2-6, 2008, Long Beach, CA, USA},
      journal = {Biophys. J.},
      year = {2008},
      volume = {94},
      pages = {659},
      url = {http://www.cell.com/biophysj/fulltext/S0006-3495%2808%2979117-3}
    }
    
    Esteban-Martín, S., Strandberg, E., Ulrich, A.S. & Salgado, J. Nonambiguous Orientation and Dynamics of Membrane Peptides from PISEMA NMR Spectra 2008 Biophys. J.
    Vol. 94 (2, S), Biophysical Society 52th Annual Meeting & 16th International Biophysics Congress, February 2-6, 2008, Long Beach, CA, USA, p. 260 
    Conference URL 
    Abstract: In the smectic phase of liquid crystal lipid bilayers, embedded peptides exhibit orientational order which can be characterized by a number of spectroscopic methods. Molecular dynamics (MD) simulations suggest that such a peptide orientation is highly fluctuating and the corresponding dynamics affects experimental measures, like 2H NMR splittings (S. Esteban-Martin and J. Salgado, Biophys J., in press). In this work, we investigate the influence of broad distributions of the orientational parameters, tilt and polarity (self-axis rotation), of helical peptides in membranes, on 1H-15N dipolar couplings and 15N CSA. Both observables are usually registered simultaneously as 2D PISEMA spectra, where they form wheel patterns from which the tilt and self-rotation of the peptide can be determined. We calculate NMR parameters from idealized peptide systems with Gaussian fluctuations of the tilt and self-rotation (no structural fluctuations allowed), and find that such a peptide whole- body dynamics affects the simulated PISEMA spectra. However, as a difference with respect to the 2H NMR splittings, nonambiguous orientations can still be determined, additionally yielding the standard deviation of the corresponding orientational distributions. For reported experimental PISEMA of oligomeric peptides, a re-analysis including dynamics gives tilt and rotation angles comparable with the values from a static model, although accompanied by distributions of a small width. In contrast, theoretical spectra expected for highly dynamic monomeric peptides cannot be described with a static model, although a dynamic model provides both, the orientational parameters and the width of their distributions. Experimental measurements on the WLP23 model peptide, thought to be monomeric, are being performed to test our predictions.
    BibTeX:
    @conference{Esteban2008,
      author = {Santiago Esteban-Martín and Erik Strandberg and Anne S. Ulrich and Jesús Salgado},
      title = {Nonambiguous Orientation and Dynamics of Membrane Peptides from PISEMA NMR Spectra},
      booktitle = {Biophysical Society 52th Annual Meeting & 16th International Biophysics Congress, February 2-6, 2008, Long Beach, CA, USA},
      journal = {Biophys. J.},
      year = {2008},
      volume = {94},
      pages = {260},
      url = {http://www.cell.com/biophysj/fulltext/S0006-3495%2808%2979029-5}
    }
    
    Rodrigo, G., Montagud, A., Aparici, A., Aroca, M., Baguena, M., Carrera, J., Edo, C., Fernandez-de-Cordoba, P., Ferrando, A., Fuertes, G., Gimenez, D., Mata, C., Medrano, J., Navarrete, C., Navarro, E., Salgado, J., Tortosa, P., Urchueguia, J. & Jaramillo, A. Vanillin cell sensor 2007 IET Synth. Biol.
    Vol. 1(1-2), pp. 74-78 
    Article DOI
    Citations 
    Abstract: Our project for iGEM 2006 consisted of designing a cellular vanillin biosensor. We used an EnvZ–E. coli strain as a chassis, and constructed two different devices: a sensor and an actuator, assembled using OmpR-P as a standardised mediator. The sensor device contained a computationally designed vanillin receptor and a synthetic two-component signal transduction protein (Trz). The receptor protein was based on a ribose-binding protein as scaffold. The Trz was built by fusion of the periplasmic and transmembrane domains of a Trg protein with an EnvZ kinase domain. When the receptor complex binds Trg, an allosteric motion is propagated to the cytoplasmic EnvZ kinase domain, resulting in autophosphorylation and subsequent phosphate transfer to the OmpR transcription factor, which finally induces transcription of the ompC promoter. As actuator, we used a synthetic transcriptional circuit, which implements an OmpR-P band detector having GFP and RFP as an output. We designed this circuit using a synthetic promoter working as an AND gate, which is synergistically activated by cI and CRP. Our constructed Trg-EnvZ fusion and AND promoter will be very useful to future synthetic biology projects.
    BibTeX:
    @article{Rodrigo2007,
      author = {G. Rodrigo and A. Montagud and A. Aparici and M.C. Aroca and M. Baguena and J. Carrera and C. Edo and P. Fernandez-de-Cordoba and A. Ferrando and G. Fuertes and D. Gimenez and C. Mata and J.V. Medrano and C. Navarrete and E. Navarro and J. Salgado and P. Tortosa and J. Urchueguia and A. Jaramillo},
      title = {Vanillin cell sensor},
      journal = {IET Synth. Biol.},
      year = {2007},
      volume = {1},
      number = {1-2},
      pages = {74-78},
      doi = {http://dx.doi.org/10.1049/iet-stb:20060003}
    }
    
    García-Sáez, A.J., Chiantia, S., Salgado, J. & Schwille, P. Mechanism of pore formation studied by AFM: effect of Bax-derived peptide on the line tension 2007 Biophys. J.
    Vol. S, Biophysical Society 51th Annual Meeting, March 3-7, 2007, Baltimore, Maryland, USA, pp. 612A-612A 
    Conference
    URL 
    Abstract: Bax, a regulator of apoptosis of the Bcl-2 family of proteins, is involved in the release of apoptotic factors across the outer mitochondrial membrane. We recently found that small fragments derived from Bax display poration activity similar to their parent protein. The detailed mechanism of action is unknown, but it involves the induction and stabilization of lipidic, or mixed lipidic/proteinaceous pores of toroidal structure.
    Theoretical models describe these pores as meta-stable arrangements, with free energy of formation being a function of the pore radius, r, as:
    Er0 = 2⋅ π⋅r⋅γ−π⋅r2⋅σ
    where γ is the line tension opposing the pore, related to the curvature stress at the place of monolayer fusion, and σ is the surface tension, which favours pore formation and expansion.
    To investigate the mechanism of protein-induced lipid pore formation, we used a peptide derived from helix 5 of Bax (Bax-α5), which is able to induce Bax-like pores in lipid bilayers. We used confocal microscopy and AFM to image shape changes in domain-exhibiting supported bilayers. The presence of Bax-α5 reduced the force necessary to rupture the bilayer with the AFM tip in force experiments. Our results show that Bax-α5 reduces the line tension at the boundaries of phase-separated bilayers and at the edge of membrane pores. Such a decrease in line tension may be a general strategy of pore forming peptides and proteins. It affects the energetics of the pore and stabilizes the open state.
    BibTeX:
    @conference{Garcia2007a,
      author = {A. J. Garcia-Saez and S. Chiantia and J. Salgado and P. Schwille},
      title = {Mechanism of pore formation studied by AFM: effect of Bax-derived peptide on the line tension},
      booktitle = {Biophysical Society 51th Annual Meeting, March 3-7, 2007, Baltimore, Maryland, USA},
      journal = {Biophys. J.},
      year = {2007},
      volume = {S},
      pages = {612A-612A},
      note = {PT: J; SU: Suppl. S},
      url = {http://www.biophysics.org/abstracts/}
    }
    
    García-Sáez, A.J., Chiantia, S., Salgado, J. & Schwille, P. Pore formation by a bax-derived peptide: Effect on the line tension of the membrane probed by AFM 2007 Biophys. J.
    Vol. 93(1), pp. 103-112 
    Article DOI
    Citations 
    Abstract: Bax is a critical regulator of physiological cell death that increases the permeability of the outer mitochondrial membrane and facilitates the release of the so-called apoptotic factors during apoptosis. The molecular mechanism of action is unknown, but it probably involves the formation of partially lipidic pores induced by Bax. To investigate the interaction of Bax with lipid membranes and the physical changes underlying the formation of Bax pores, we used an active peptide derived from helix 5 of this protein (Bax-alpha 5) that is able to induce Bax-like pores in lipid bilayers. We report the decrease of line tension due to peptide binding both at the domain interface in phase-separated lipid bilayers and at the pore edge in atomic force microscopy. lm-rupture experiments. Such a decrease in line tension may be a general strategy of pore-forming peptides and proteins, as it affects the energetics of the pore and stabilizes the open state.
    BibTeX:
    @article{Garcia2007,
      author = {A. J. Garcia-Saez and S. Chiantia and J. Salgado and P. Schwille},
      title = {Pore formation by a bax-derived peptide: Effect on the line tension of the membrane probed by AFM},
      journal = {Biophys. J.},
      year = {2007},
      volume = {93},
      number = {1},
      pages = {103-112},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1529/biophysj.106.100370}
    }
    
    Esteban-Martín, S. & Salgado, J. Self-assembling of peptide/membrane complexes by atomistic molecular dynamics simulations 2007 Biophys. J.
    Vol. 92(3), pp. 903-912 
    Article DOI
    Citations 
    Abstract: Model biological membranes consisting of peptide/lipid-bilayer complexes can nowadays be studied by classical molecular dynamics (MD) simulations at atomic detail. In most cases, the simulation starts with an assumed state of a peptide in a preformed bilayer, from which equilibrium configurations are difficult to obtain due to a relatively slow molecular diffusion. As an alternative, we propose an extension of reported work on the self-organization of unordered lipids into bilayers, consisting of including a peptide molecule in the initial random configuration to obtain a membrane-bound peptide simultaneous to the formation of the lipid bilayer. This strategy takes advantage of the fast reorganization of lipids, among themselves and around the peptide, in an aqueous environment. Model peptides of different hydrophobicity, CH3-CO-W2L18W2-NH2 (WL22) and CH3-CO-W2A18W2-NH2 (WA22), in dipalmitoyl-phosphatidylcholine (DPPC) are used as test cases. In the equilibrium states of the peptide/membrane complexes, achieved in time ranges of 50-100 ns, the two peptides behave as expected from experimental and theoretical studies. The strongly hydrophobic WL22 is inserted in a transmembrane configuration and the marginally apolar, alanine-based WA22 is found in two alternative states: transmembrane inserted or parallel to the membrane plane, embedded close to the bilayer interface, with similar stability. This shows that the spontaneous assembly of peptides and lipids is an unbiased and reliable strategy to produce and study models of equilibrated peptide/lipid complexes of unknown membrane-binding mode and topology.
    BibTeX:
    @article{Esteban2007a,
      author = {S. Esteban-Martin and J. Salgado},
      title = {Self-assembling of peptide/membrane complexes by atomistic molecular dynamics simulations},
      journal = {Biophys. J.},
      year = {2007},
      volume = {92},
      number = {3},
      pages = {903-912},
      note = {PUBM: Print-Electronic; DEP: 20061103; JID: 0370626; 0 (Lipid Bilayers); 0 (Macromolecular Substances); 0 (Peptides); 0 (Phospholipids); 2006/11/03 [aheadofprint]; ppublish},
      doi = {http://dx.doi.org/10.1529/biophysj.106.093013}
    }
    
    Esteban-Martín, S. & Salgado, J. The dynamic orientation of membrane-bound peptides: Bridging simulations and experiments 2007 Biophys. J.
    Vol. 93(12), pp. 4278-4288 
    Article DOI
    Citations 
    Abstract: The structural organization in a peptide/membrane supramolecular complex is best described by knowledge of the peptide orientation plus its time-dependent and spacial fluctuations. The static orientation, defined by the peptide tilt and a rotation about its molecular axis, is accessible through a number of spectroscopic methods. However, peptide dynamics, although relevant to understand the functionality of these systems, remains largely unexplored. Here, we describe the orientation and dynamics of Trp-flanked and Lys-flanked hydrophobic peptides in a lipid bilayer from molecular dynamics simulations. A novel view is revealed, where collective non-trivial distributions of time-evolving and ensemble peptide orientations closely represent the systems as studied experimentally. Such global distributions are broad and unveil the existence of orientational states, which depend on the anchoring mode of interfacial residues. We show that this dynamics modulates 2H quadrupolar splittings and introduces ambiguity in the analysis of NMR data. These findings demonstrate that structural descriptions of peptide/membrane complexes are incomplete, and in cases even imprecise, without knowledge of dynamics.
    BibTeX:
    @article{Esteban2007,
      author = {S. Esteban-Martin and J. Salgado},
      title = {The dynamic orientation of membrane-bound peptides: Bridging simulations and experiments},
      journal = {Biophys. J.},
      year = {2007},
      volume = {93},
      number = {12},
      pages = {4278-4288},
      note = {PUBM: Print-Electronic; DEP: 20070824; JID: 0370626; aheadofprint},
      doi = {http://dx.doi.org/10.1529/biophysj.107.113043}
    }
    
    Fuertes, G., Schlemmer, C. & Salgado, J. Correlation between the aggregation behaviour of Bcl-xL and its membrane-active properties 2007 Eur. Biophys. J.
    Vol. 36 (Suppl 1):S51-S248, 6th EBSA & British Biophysical Society Congress, July 14-18, 2007, Imperial College, London, U.K., pp. P-173 
    Conference DOI 
    Abstract: The anti-apoptotic protein Bcl-xL, a member of the Bcl-2 family, performs its function at the outer mitochondrial membrane where it inhibits pro-apoptotic counterparts through unknown mechanisms. Pro-apoptotic proteins of the same family, like Bax, possess poreforming capability permitting the release of apoptotic factors from mitochondria. A pair of central amphipathic α-helices, α5α6, appears to be the minimum motif needed to permeabilize membranes. This domain is also present in Bcl-xL with a high level of homology with that of Bax-type proteins. We have studied both full-length Bcl-xL and fragments derived from the central helices. We show that Bcl-xL has also membrane-destabilizing effects and is able to release small probes encapsulated into model membrane systems but only at acidic pH. In parallel, we have estimated the size of the proteins at different pHs. According to light scattering measurements, Bcl-xL is dimeric at neutral pH, but associates into large aggregates at acidic pH. In contrast, Bcl-xL α5α6 is already aggregated at pH 7 and oligomerizes further as the pH is lowered. These proteins seem to partition into liposomes only at acidic pH and the size of vesicles upon addition of proteins remains unaffected. Taken together these results suggest that Bcl-xL is able to form pores of moderate size in lipid bilayers at acidic pH and the acquisition of an oligomeric structure may be involved in the pore-forming activity. On the other hand, at neutral pH, Bcl-xL does not cause leakage from vesicles, probably because a positive net charge in the protein is required for an appropriate interaction with the membrane lipids.
    BibTeX:
    @conference{Fuertes2007,
      author = {Fuertes, G. and Schlemmer, C. and Salgado, J.},
      title = {Correlation between the aggregation behaviour of Bcl-xL and its membrane-active properties},
      booktitle = {6th EBSA & British Biophysical Society Congress, July 14-18, 2007, Imperial College, London, U.K.},
      journal = {Eur. Biophys. J.},
      year = {2007},
      volume = {36 (Suppl 1):S51-S248},
      pages = {P-173},
      doi = {http://dx.doi.org/10.1007/s00249-007-0178-7}
    }
    
    Lukovic, D., Plasencia, I., Taberner, F.J., Salgado, J., Calvete, J.J., Perez-Gil, J. & Mingarro, I. Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity 2006 Biochim. Biophys. Acta, Biomembr.
    Vol. 1758(4), pp. 509-518 
    Article DOI
    Citations 
    Abstract: Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% alpha-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression pi-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.
    BibTeX:
    @article{Lukovic2006,
      author = {D. Lukovic and I. Plasencia and F. J. Taberner and J. Salgado and J. J. Calvete and J. Perez-Gil and I. Mingarro},
      title = {Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity},
      journal = {Biochim. Biophys. Acta, Biomembr.},
      year = {2006},
      volume = {1758},
      number = {4},
      pages = {509-518},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1016/j.bbamem.2006.03.005}
    }
    
    García-Sáez, A.J., Coraiola, M., Serra, M.D., Mingarro, I., Muller, P. & Salgado, J. Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid pores 2006 FEBS J.
    Vol. 273(5), pp. 971-981 
    Article DOI
    Citations 
    Abstract: Proteins of the B-cell lymphoma protein 2 (Bcl2) family are key regulators of the apoptotic cascade, controlling the release of apoptotic factors from the mitochondrial intermembrane space. A helical hairpin found in the core of water-soluble folds of these proteins has been reported to be the pore-forming domain. Here we show that peptides including any of the two alpha-helix fragments of the hairpin of Bcl2 associated protein X (Bax) can independently induce release of large labelled dextrans from synthetic lipid vesicles. The permeability promoted by these peptides is influenced by intrinsic monolayer curvature and accompanied by fast transbilayer redistribution of lipids, supporting a toroidal pore mechanism as in the case of the full-length protein. However, compared with the pores made by complete Bax, the pores made by the Bax peptides are smaller and do not need the concerted action of tBid. These data indicate that the sequences of both fragments of the hairpin contain the principal physicochemical requirements for pore formation, showing a parallel between the permeabilization mechanism of a complex regulated protein system, such as Bax, and the much simpler pore-forming antibiotic peptides.
    BibTeX:
    @article{Garcia2006,
      author = {A. J. Garcia-Saez and M. Coraiola and M. D. Serra and I. Mingarro and P. Muller and J. Salgado},
      title = {Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid pores},
      journal = {FEBS J.},
      year = {2006},
      volume = {273},
      number = {5},
      pages = {971-981},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1111/j.1742-4658.2006.05123.x}
    }
    
    Sauri, A., Saksena, S., Salgado, J., Johnson, A.E. & Mingarro, I. Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted 2005 J. Biol. Chem.
    Vol. 280(27), pp. 25907-25912 
    Article DOI
    Citations 
    Abstract: The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.
    BibTeX:
    @article{Sauri2005,
      author = {A. Sauri and S. Saksena and J. Salgado and A. E. Johnson and I. Mingarro},
      title = {Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted},
      journal = {J. Biol. Chem.},
      year = {2005},
      volume = {280},
      number = {27},
      pages = {25907-25912},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1074/jbc.M412476200}
    }
    
    García-Sáez, A.J. Characterising the interactions of fragments derived from Bcl-xL, Bax and Bid with lipid membranes. 2005 PhD Thesis,
    University of Valencia, Burjassot, Spain. 
    PhD Thesis URL 
    Abstract:
    BibTeX:
    @phdthesis{Garcia2005b,
      address = {Spain},
      type = {{PhD}},
      author = {Ana J. García-Sáez},
      title = {Characterising the interactions of fragments derived from Bcl-xL, Bax and Bid with lipid membranes},
      School = {University of Valencia},
      language = {English}
      year = {2005},
      url = {https://www.educacion.gob.es/teseo/mostrarRef.do?ref=379599}
    }
    
    García-Sáez, A.J., Coraiola, M., Serra, M.D., Mingarro, I., Menestrina, G. & Salgado, J. Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins 2005 Biophys. J.
    Vol. 88(6), pp. 3976-3990 
    Article DOI
    Citations 
    Abstract: Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first alpha-helix of a colicinlike hydrophobic hairpin (alpha-helix 5 of Bax and alpha-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly alpha-helical structures and induce the release of entrapped calcein. In planar lipid membranes they form ion conducting channels, which in the case of the Bax-derived peptide are characterized by a two-stage pattern, a large conductivity and lipid-charge-dependent ionic selectivity. These features, together with the influence of intrinsic lipid curvature on the poration activity and the existence of two helical stretches of different orientations for the membrane-bound peptide, suggest that it forms mixed lipidic/peptidic pores of toroidal structure. In contrast, the assayed Bid fragment shows a markedly different behavior, characterized by the formation of discrete, steplike channels in planar lipid bilayers, as expected for a peptidic pore lined by a bundle of helices.
    BibTeX:
    @article{Garcia2005,
      author = {A. J. García-Sáez and M. Coraiola and M. Dalla Serra and I. Mingarro and G. Menestrina and J. Salgado},
      title = {Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins},
      journal = {Biophys. J.},
      year = {2005},
      volume = {88},
      number = {6},
      pages = {3976-3990},
      note = {LR: 20061115; PUBM: Print-Electronic; DEP: 20050318; JID: 0370626; 0 (BAX protein, human); 0 (BH3 Interacting Domain Death Agonist Protein); 0 (BID protein, human); 0 (Carrier Proteins); 0 (Ion Channels); 0 (Liposomes); 0 (Multiprotein Complexes); 0 (Peptide Fragments); 0 (Proto-Oncogene Proteins c-bcl-2); 0 (bcl-2-Associated X Protein); 2005/03/18 [aheadofprint]; 2005/03/25 [aheadofprint]; ppublish},
      doi = {http://dx.doi.org/10.1529/biophysj.104.058008}
    }
    
    Pérez-Payá, E., García-Sáez, A.J., Malet, G., Mingarro, I. & Salgado, J. Modulation of protein-membrane and protein-protein interactions in apoptotic pathways 2004 Protein Sci.
    Vol. 13, 18th Symposium of the Protein Society, August 14-18, 2004, San Diego, CA, USA, pp. 199-200 
    Conference
     
    BibTeX:
    @conference{Perez-P2004,
      author = {E. Perez-Paya and A. J. Garcia-Saez and G. Malet and I. Mingarro and J. Salgado},
      title = {Modulation of protein-membrane and protein-protein interactions in apoptotic pathways},
      booktitle = {18th Symposium of the Protein Society, August 14-18, 2004, San Diego, CA, USA},
      journal = {Protein Sci.},
      year = {2004},
      volume = {13},
      pages = {199-200},
      note = {PT: J; SU: Suppl. 1}
    }
    
    Orzaez, M., Salgado, J., Gimenez-Giner, A., Pérez-Payá, E. & Mingarro, I. Influence of proline residues in transmembrane helix packing 2004 J. Mol. Biol.
    Vol. 335(2), pp. 631-640 
    Article DOI
    Citations 
    Abstract: Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In addition, both glycosylation mapping in biological membranes and molecular dynamics showed that the presence of a proline residue at the lipid/water interface has as an effect the extension of the helical end. Thus, helix packing can be an important factor that determines appearance of proline in TM helices. Membrane proteins might accumulate proline residues at the two ends of their TM segments in order to modulate the exposition of key amino acid residues at the interface for molecular recognition events while allowing stable association and native folding.
    BibTeX:
    @article{Orzaez2004,
      author = {M. Orzaez and J. Salgado and A. Gimenez-Giner and E. Perez-Paya and I. Mingarro},
      title = {Influence of proline residues in transmembrane helix packing},
      journal = {J. Mol. Biol.},
      year = {2004},
      volume = {335},
      number = {2},
      pages = {631-640},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1016/j.jmb.2003.10.062}
    }
    
    García-Sáez, A.J., Mingarro, I., Pérez-Payá, E. & Salgado, J. Membrane-insertion fragments of Bcl-xL, Bax, and Bid 2004 Biochemistry
    Vol. 43(34), pp. 10930-10943 
    Article DOI
    Citations 
    Abstract: Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapoptotic Bcl-x(L) and proapoptotic Bax and Bid, that correspond to defined alpha helices in the structure of their soluble forms. According to prediction methods, there are only two putative TM fragments in Bcl-x(L) and Bax (the C-terminal alpha helix and alpha-helix 5) and one in activated tBid (alpha-helix 6). The rest of their sequence, including the second helix of the pore-forming domain, displays only weak hydrophobic peaks, which are below the prediction threshold. Subsequent analysis by glycosylation mapping of single alpha-helix segments in a model chimeric system confirms the above predictions and allows finding an extra TM fragment made of helix alpha1 of Bax. Surprisingly, the amphipathic helices alpha6 of Bcl-x(L) and Bax and alpha7 of Bid do insert in membranes only as part of the alpha5-alpha6 (Bcl-x(L) and Bax) or alpha6-alpha7 (Bid) hairpins but not when assayed individually. This behavior suggests a synergistic insertion and folding of the two helices of the hairpin that could be due to charge complementarity and additional stability provided by turn-inducing residues present at the interhelical region. Although these data come from chimeric systems, they show direct potentiality for acquiring a membrane inserted state. Thus, the above fragments should be considered for the definition of plausible models of the active, membrane-bound species of Bcl-2 proteins.
    BibTeX:
    @article{Garcia2004,
      author = {A. J. Garcia-Saez and I. Mingarro and E. Perez-Paya and J. Salgado},
      title = {Membrane-insertion fragments of Bcl-xL, Bax, and Bid},
      journal = {Biochemistry},
      year = {2004},
      volume = {43},
      number = {34},
      pages = {10930-10943},
      note = {LR: 20061115; PUBM: Print; JID: 0370623; 0 (BAX protein, human); 0 (BCL2L1 protein, human); 0 (BH3 Interacting Domain Death Agonist Protein); 0 (BID protein, human); 0 (Bax protein, mouse); 0 (Bcl2l1 protein, mouse); 0 (Bid protein, mouse); 0 (Carrier Proteins); 0 (Membrane Lipids); 0 (Peptide Fragments); 0 (Proto-Oncogene Proteins); 0 (Proto-Oncogene Proteins c-bcl-2); 0 (Recombinant Fusion Proteins); 0 (bcl-2-Associated X Protein); 0 (bcl-X Protein); ppublish},
      doi = {http://dx.doi.org/10.1021/bi036044c}
    }
    
    Ulrich, A.S., Afonin, S.E., Glaser, R.W., Duerr, U., Salgado, J., Grage, S.L., Berditchevskaia, M. & Wadhwani, P. NMR studies of antimicrobial and fusogenic peptides in membranes 2002 Biophys. J.
    Vol. 82(1), Biophysical Society 46th Annual Meeting, 2002, pp. 514A-514A 
    Conference
    URL 
    Abstract: The alignment and dynamic behavior of several membrane-active peptides in lipid bilayers was determined by solid state NMR, based on angular and distance constraints in oriented samples. We employ 19F as a reporter, as it is more sensitive than conventional NMR-labels and can address longer-range distances. The peptides were substituted at selected hydrophobic positions with 4F-phenylglycine, usually without disturbing the conformation or biological activity. Here, we compare two structurally distinct antimicrobial peptides, which destroy bacteria by destabilizing their cell membranes: gramicidin S is a cyclic beta-sheet, while "PGLa" is alpha-helical. A third peptide, "B18", can trigger rapid fusion between lipid vesicles and has a helix-loop-helix structure. The behavior of gramicidin S was found to depend strongly on peptide concentration and temperature, whereas PGLa responds smoothly to these parameters. Both antimicrobial peptides are preferentially aligned with their amphiphilic face parallel to the membrane plane, but gramicidin S appears to re-arrange in its functionally active state into an oligomeric pore. The fusogenic peptide, on the other hand, aggregates as inactive amyloid fibrils at high concentration, while at low concentration it assumes a uniform structure and penetrates the bilayer at an oblique angle.
    BibTeX:
    @conference{Ulrich2002,
      author = {A. S. Ulrich and S. E. Afonin and R. W. Glaser and U. Duerr and J. Salgado and S. L. Grage and M. Berditchevskaia and P. Wadhwani},
      title = {NMR studies of antimicrobial and fusogenic peptides in membranes},
      booktitle = {Biophysical Society 46th Annual Meeting, 2002},
      journal = {Biophys. J.},
      year = {2002},
      volume = {82},
      number = {1},
      pages = {514A-514A},
      note = {PT: J; PN: Part 2},
      url = {http://www.biophysics.org/abstracts/}
    }
    
    Salgado, J., García-Sáez, A.J., Malet, G., Mingarro, I. & Pérez-Payá, E. Peptides in apoptosis research 2002 J. Pept. Sci.
    Vol. 8(10), pp. 543-560 
    Article DOI
    Citations 
    Abstract: Apoptosis is a complex process that plays a central role in physiological and pathological cell death. This fast evolving research area has experienced incredible development in the past few years. Progress in the knowledge of the structure of many of the main molecular actors of the apoptotic signal transduction pathways has driven the design of synthetic peptides that in some cases can function as simplified versions of their parent proteins. These molecules are contributing to a better understanding of the activity and regulation of apoptotic proteins and also are setting the basis for the discovery of effective drugs to combat important diseases related to apoptosis. Most applications of peptides in apoptosis research are so far related to caspases, caspase regulatory proteins, such as IAPs and Smac, and proteins of the Bcl-2 family. Additionally, important perspectives are open to other systems, such as the macromolecular assemblies that are responsible for the activation of initiator caspases.
    BibTeX:
    @article{Salgado2002,
      author = {J. Salgado and A. J. Garcia-Saez and G. Malet and I. Mingarro and E. Perez-Paya},
      title = {Peptides in apoptosis research},
      journal = {J. Pept. Sci.},
      year = {2002},
      volume = {8},
      number = {10},
      pages = {543-560},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1002/psc.414}
    }
    
    Grage, S.L., Afonin, S.E., Salgado, J., Duerr, U., Cross, T.A. & Ulrich, A.S. Flourine solid state NMR on biomembranes 2002 Biophys. J.
    Vol. 82(1), Biophysical Society 46th Annual Meeting, 2002, pp. 519A-519A 
    Conference
    URL 
    Abstract: Solid State NMR is a powerful method to obtain structural information on the atomic level and characterize dynamics in biomembranes. This technique is, however, limited by the low sensitivity and short distance range of conventionally used NMR labels such as 13C, 2H or 15N. To overcome these limitations we propose 19F as an NMR probe in biomembranes. We developed a 19F solid state NMR strategy which is based on the use of oriented membranes, and the analysis of 19F-NMR lineshapes obtained at different sample tilts in the magnetic field. This way, structural information was gained from the anisotropy of the chemical shift and 19F-19F dipolar couplings. We improved the Carr-Purcell- Meiboom-Gill experiment to measure homonuclear dipolar couplings without the need of magic angle spinning or 1H-decoupling. This novel approach was developed with the aid of 19F-labelled lipid soluble drugs, where dipolar couplings between 2, 3 and 6 spins could be obtained, and conclusions on the behaviour of the guest molecules in the membrane were drawn from lineshape analysis of

    the dipolar spectra. The approach was extended to the antimicrobial peptides gramicidin

    A and gramicidin S, labelled with F-tryptophan and F-phenylglycine. On the basis of our 19F NMR results we were able to propose a pore mechanism for the antimicrobial action of the cyclic β-sheet gramicidin S.

    BibTeX:
    @conference{Grage2002,
      author = {S. L. Grage and S. E. Afonin and J. Salgado and U. Duerr and T. A. Cross and A. S. Ulrich},
      title = {Flourine solid state NMR on biomembranes},
      booktitle = {Biophysical Society 46th Annual Meeting, 2002},
      journal = {Biophys. J.},
      year = {2002},
      volume = {82},
      number = {1},
      pages = {519A-519A},
      note = {PT: J; PN: Part 2},
      url = {http://www.biophysics.org/abstracts/}
    }
    
    Salgado, J., Grage, S.L., Kondejewski, L.H., Hodges, R.S., McElhaney, R.N. & Ulrich, A.S. Membrane-bound structure and alignment of the antimicrobial beta-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR 2001 J. Biomol. NMR
    Vol. 21(3), pp. 191-208 
    Article DOI
    URL
    Citations 
    Abstract: The antimicrobial properties of the cyclic beta -sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state F-19-NMR was used to study a F-19-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two F-19-labels was measured from their homonuclear dipolar coupling as 6 Angstrom, in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the F-19-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the F-19-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic beta -sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the F-19-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the F-19 nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.
    BibTeX:
    @article{Salgado2001,
      author = {J. Salgado and S. L. Grage and L. H. Kondejewski and R. S. Hodges and R. N. McElhaney and A. S. Ulrich},
      title = {Membrane-bound structure and alignment of the antimicrobial beta-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR},
      journal = {J. Biomol. NMR},
      year = {2001},
      volume = {21},
      number = {3},
      pages = {191-208},
      note = {LR: 20061115; PUBM: Print; JID: 9110829; 0 (Anti-Bacterial Agents); 0 (Lipid Bilayers); 0 (Membrane Proteins); 13699-48-4 (Dimyristoylphosphatidylcholine); 1405-97-6 (Gramicidin); 7782-41-4 (Fluorine); ppublish},
      url = {http://www.springerlink.com/content/t253437677345748/fulltext.pdf},
      doi = {http://dx.doi.org/10.1023/A:1012946026231}
    }
    
    Grage, S.L., Salgado, J., Dürr, U.H.N., Afonin, S.E., Glaser, R.W. & Ulrich, A.S. Solid State 19F-NMR of Biomembranes 2001 In Perspectives on Solid State NMR in Biology
    (series: Focus on Structural Biology, vol. 1), Kiihne, S. & de Groot, H. (eds.). Kluwer Academic Publishers, Dordrecht, pp. 83-91.
    Chapter URL
    Citations 
    BibTeX:
    @Chapter{Grage2001,
      author = {Grage, S. L. and Salgado, J. and Dürr, U. H. N. and Afonin, S. E. and Glaser, R. W. and Ulrich, A. S.},
      title = {Solid State 19F-NMR of Biomembranes},
      booktitle = {Perspectives on Solid State NMR in Biology},
      series = {Focus on Structural Biology},
      editor = {Kiihne, S.R. and de Groot, H.J.M.},
      publisher = {Kluwer Academic Publishers},
      address = {Dordrecht},
      year = {2001},
      volume = {1},
      pages = {83-91},
      url = {href="http://link.springer.com/chapter/10.1007/978-94-017-2579-8_8}
    }
    
    Bubacco, L., Vijgenboom, E., Gobin, C., Tepper, A.W.J.W., Salgado, J. & Canters, G.W. Kinetic and paramagnetic NMR investigations of the inhibition of Streptomyces antibioticus tyrosinase 2000 J. Mol. Catal., B Enzym.
    Vol. 8(1-3), pp. 27-35 
    Article DOI
    Citations 
    Abstract: A scaled-up isolation and purification procedure is described for tyrosinase from Streptomyces antibioticus. Kinetic studies of the enzyme catalysed conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) into DOPAchrome show that kojic acid, L-mimosine, p-toluic acid and benzoic acid exhibit competitive inhibition with inhibition constants of 3.4, 30, 1.9 X 10(2) and 8.0 X 10(2) mu M, respectively. Paramagnetic NMR techniques appear well suited to study the binding of inhibitors to the active site. From the variation of the NMR shifts with temperature a value of -2J = 156 +/- 6 cm(-1) is derived for the exchange coupling between the unpaired spins on the two Cu(II) ions in the active site of the met-tyrosinase/kojic acid complex. (C) 2000 Elsevier Science B.V. All rights reserved.
    BibTeX:
    @article{Bubacco2000,
      author = {L. Bubacco and E. Vijgenboom and C. Gobin and A. W. J. W. Tepper and J. Salgado and G. W. Canters},
      title = {Kinetic and paramagnetic NMR investigations of the inhibition of Streptomyces antibioticus tyrosinase},
      journal = {J. Mol. Catal., B Enzym.},
      year = {2000},
      volume = {8},
      number = {1-3},
      pages = {27-35},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1016/S1381-1177(99)00064-8}
    }
    
    Salgado, J., Kalverda, A.P., Diederix, R.E.M., Canters, G.W., Moratal, J.M., Lawler, A.T. & Dennison, C. Paramagnetic NMR investigations of Co(II) and Ni(II) amicyanin 1999 J. Biol. Inorg. Chem.
    Vol. 4(4), pp. 457-467 
    Article DOI
    Citations 
    Abstract: The paramagnetic H-1 NMR spectra of the Co(II) and Ni(II) substituted forms of the type 1 blue copper protein (cupredoxin) amicyanin have been assigned. This is the first such analysis of a cupredoxin, which has a distorted tetrahedral active site with the ligands provided by two histidines, a cysteine and a methionine. The isotropic shifts of the resonances in these spectra are compared with those of Co(II) and Ni(II) azurin. A number of interesting similarities and differences are found. The coordination of the metal by the two equatorial histidine ligands is very similar in both proteins. The interaction between the introduced metal and the thiolate sulfur of the equatorial cysteine ligand is enhanced in the amicyanin derivatives. Resonances belonging to the weak axial methionine ligand exhibit much larger shifts in the amicyanin derivatives, indicative of shorter M(II)-S(Met) distances. The presence of shorter axial M(II)-S(Met) and equatorial M(II)-S(Cys) distances in both Co(II) and Ni(II) amicyanin is ascribed to the absence of a second axially interacting amino acid at the active site of this cupredoxin.
    BibTeX:
    @article{Salgado1999,
      author = {J. Salgado and A. P. Kalverda and R. E. M. Diederix and G. W. Canters and J. M. Moratal and A. T. Lawler and C. Dennison},
      title = {Paramagnetic NMR investigations of Co(II) and Ni(II) amicyanin},
      journal = {J. Biol. Inorg. Chem.},
      year = {1999},
      volume = {4},
      number = {4},
      pages = {457-467},
      note = {PT: J},
      doi = {href="http://dx.doi.org/10.1007/s007750050332}
    }
    
    Kolczak, U., Salgado, J., Siegal, G., Saraste, M. & Canters, G.W. Paramagnetic NMR studies of blue and purple copper proteins 1999 Biospectroscopy
    Vol. 5(5), pp. S19-S32 
    Article DOI
    Citations 
    Abstract: H-1- and C-13-NMR spectroscopy is applied to investigate the Cu, and type 1 active sites of copper proteins in solution. The analysis of hyperfine shifted H-1 resonances allows the comparison of the electron spin density delocalization in the Cu-A site of the wild-type soluble domains of various cytochrome c oxidases (Thermus thermophilus, Paracoccus denitrificans, and Paracoccus versutus) and genetically engineered constructs (soluble domain of quinol oxidase from Escherichia coli and Thiobacillus versutus amicyanin). Comparable spin densities are found on the two terminal His ligands for the wild-type constructs as opposed to the engineered proteins where the spin is more unevenly distributed on the two His residues. A reevaluation of the Cys H-beta chemical shifts that is in agreement with the data published for both the P. denitrificans and the P. versutus Cu-A soluble domains confirms the thermal accessibility of the B-2(3u) electronic excited state and indicates the existence of slightly different spin densities on the two bridging Cys ligands. The C-13-NMR spectrum of isotopically enriched oxidized azurin from Pseudomonas aeruginosa reveals six fast relaxing signals, which can be partially identified by 1- and 2-dimensional (1-D, 2-D) direct detection techniques combined with 3-D triple resonance experiments. The observed contact shifts suggest the presence of direct spin density transfer and spin polarization mechanisms for the delocalization of the unpaired electron.
    BibTeX:
    @article{Kolczak1999,
      author = {U. Kolczak and J. Salgado and G. Siegal and M. Saraste and G. W. Canters},
      title = {Paramagnetic NMR studies of blue and purple copper proteins},
      journal = {Biospectroscopy},
      year = {1999},
      volume = {5},
      number = {5},
      pages = {S19-S32},
      note = {PT: J; SU: Suppl. S},
      url = {href="http://dx.doi.org/10.1002/(SICI)1520-6343(1999)5:5+3.0.CO;2-H}
    }
    
    Bubacco, L., Salgado, J., Tepper, A.W.J.W., Vijgenboom, E. & Canters, G.W. H-1 NMR spectroscopy of the binuclear Cu(II) active site of Streptomyces antibioticus tyrosinase 1999 FEBS Lett.
    Vol. 442(2-3), pp. 215-220 
    Article DOI
    Citations 
    Abstract: The 600 MHz H-1 NMR spectrum of tyrosinase (31 kDa) of Streptomyces antibioticus in the oxidized, chloride-bound form is reported, The downfield part of the spectrum (15-55 ppm) exhibits a large number of paramagnetically shifted signals. The paramagnetism is ascribed to a thermally populated triplet state. The signals derive from six histidines binding to the metals through their NE atoms. There is no evidence for endogenous bridges. The exchange coupling, -2J, amounts to 298 cm(-1). In the absence of chloride the peaks broaden. This is ascribed to a slowing down of the electronic relaxation. The exchange coupling decreases to -2J = 103 cm(-1) (C) 1999 Federation of European Biochemical Societies.
    BibTeX:
    @article{Bubacco1999,
      author = {L. Bubacco and J. Salgado and A. W. J. W. Tepper and E. Vijgenboom and G. W. Canters},
      title = {H-1 NMR spectroscopy of the binuclear Cu(II) active site of Streptomyces antibioticus tyrosinase},
      journal = {FEBS Lett.},
      year = {1999},
      volume = {442},
      number = {2-3},
      pages = {215-220},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1016/S0014-5793(98)01662-7}
    }
    
    Salgado, J., Warmerdam, G., Bubacco, L. & Canters, G.W. Understanding the electronic properties of the CuA site from the soluble domain of cytochrome c oxidase through paramagnetic 1H NMR 1998 Biochemistry
    Vol. 37(20), pp. 7378-7389 
    Article DOI
    Citations 
    Abstract: The soluble domain of the subunit II of cytochrome c oxidase from Paracoccus versutus was cloned, expressed, and studied by 1H NMR at 600 MHz. The properties of the redox-active dinuclear CuA site in the paramagnetic mixed-valence Cu(I)-Cu(II) state were investigated in detail. A group of relatively sharp signals found between 30 and 15 ppm in the 1H NMR spectrum correspond to the imidazole protons of the coordinated histidines (H181 and H224). A second group of broader and farther shifted signals between 50 and 300 ppm are assigned to Hbeta protons of the bridging cysteines (C216 and C220); the protons from the weak M227 and E218 ligands do not shift outside of the diamagnetic envelope. About 40% of the total spin density appears delocalized over the cysteine-bridging ligands while a much smaller amount is delocalized on the two ligand histidines. The latter have similar spin density distributions. Analysis of the pattern of the hyperfine shifts of the Cys H beta protons shows that the ground state bears 2B3u character, in which the sulfur lobes in the singly occupied molecular orbital are aligned with the Cu-Cu axis. Analysis of the temperature dependence of the shifts of the Cys H beta signals leads to the conclusion that the 2B2u excited state is thermally accessible at room temperature (Delta E approximately kT).
    BibTeX:
    @article{Salgado1998a,
      author = {J. Salgado and G. Warmerdam and L. Bubacco and G. W. Canters},
      title = {Understanding the electronic properties of the CuA site from the soluble domain of cytochrome c oxidase through paramagnetic 1H NMR},
      journal = {Biochemistry},
      year = {1998},
      volume = {37},
      number = {20},
      pages = {7378-7389},
      note = {LR: 20061115; PUBM: Print; JID: 0370623; 0 (Protons); 7440-50-8 (Copper); EC 1.9.3.1 (Electron Transport Complex IV); ppublish},
      doi = {href="http://dx.doi.org/10.1021/bi9728598}
    }
    
    Salgado, J., Kroes, S.J., Berg, A., Moratal, J.M. & Canters, G.W. The dynamic properties of the M121H azurin metal site as studied by NMR of the paramagnetic Cu(II) and Co(II) metalloderivatives 1998 J. Biol. Chem.
    Vol. 273(1), pp. 177-185 
    Article DOI
    Citations 
    Abstract: The M121H azurin mutant in solution presents various species in equilibrium that can be detected and studied by H-1 NMR of the Cu(II) and Co(II) paramagnetic metalloderivatives. In both cases up to three species are observed in slow exchange, the proportions of which are different for the two metalloderivatives. Above pH 5 the major species displays a tetrahedral coordination in which the His(121) can be observed as a coordinated residue, Its metal site corresponds to a new type of site that is defined as a type 1.5 site. The second and third species resemble the wild type (type 1) azurin and, above pH 4.5, they are present only at a low concentration. At low pH a protonation process increases the proportion of both type 1 species at the expense of the type 1.5 species. This process, characterized by a pK(a) = 4.3, is assigned to the protonation of His(121). At high pH the NMR spectrum of the Co(II)-M121H azurin experiences an additional transition, which is not observed in the case of the Cu(II) protein. The dynamic properties of the M121H metal site appear to be related to changes in the coordination geometry and the strength of the axial interaction between the N-delta 1 (His(121)) and the metal.
    BibTeX:
    @article{Salgado1998,
      author = {J. Salgado and S. J. Kroes and A. Berg and J. M. Moratal and G. W. Canters},
      title = {The dynamic properties of the M121H azurin metal site as studied by NMR of the paramagnetic Cu(II) and Co(II) metalloderivatives},
      journal = {J. Biol. Chem.},
      year = {1998},
      volume = {273},
      number = {1},
      pages = {177-185},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1074/jbc.273.1.177}
    }
    
    Donaire, A., Salgado, J. & Moratal, J.M. Determination of the magnetic axes of cobalt(II) and nickel(II) azurins from 1H NMR data: influence of the metal and axial ligands on the origin of magnetic anisotropy in blue copper proteins 1998 Biochemistry
    Vol. 37(24), pp. 8659-8673 
    Article DOI
    Citations 
    Abstract: The orientation and the axial, Deltachiax, and rhombic, Deltachirh, components of the magnetic susceptibility tensor anisotropy for the cobalt(II) and nickel(II) derivatives of azurin from Pseudomonas aeruginosa have been determined from 1H NMR data. For both derivatives, the axial geometry of the system determines the orientation of the chi-tensor, whose z-axis forms an angle of 18.6 and 20.1 degrees with the Cu-OGly45 axial bond in the cobalt(II) and nickel(II) derivatives, respectively. For protons close to this axis, large negative pseudocontact shifts are observed, while those close to the NNS plane of the equatorial ligands experience lower and positive pseudocontact shifts for the same distance. Dipolar shifts are larger in the cobalt derivative, not only because of the larger spin number but also due to its intrinsically higher anisotropy. The contact contribution to the hyperfine shifts for the coordinated residues has been evaluated and analyzed in terms of unpaired spin delocalization mechanisms and geometry considerations. The results are extended to other blue copper proteins whose cobalt derivatives have been studied by 1H NMR. The electronic structure and its implications in the redox properties of the native copper proteins are also commented.
    BibTeX:
    @article{Donaire1998,
      author = {A. Donaire and J. Salgado and J. M. Moratal},
      title = {Determination of the magnetic axes of cobalt(II) and nickel(II) azurins from 1H NMR data: influence of the metal and axial ligands on the origin of magnetic anisotropy in blue copper proteins},
      journal = {Biochemistry},
      year = {1998},
      volume = {37},
      number = {24},
      pages = {8659-8673},
      note = {LR: 20061115; PUBM: Print; JID: 0370623; 0 (Ligands); 12284-43-4 (Azurin); 7440-02-0 (Nickel); 7440-48-4 (Cobalt); ppublish},
      doi = {href="http://dx.doi.org/10.1021/bi971974f}
    }
    
    Salgado, J., Kalverda, A.P. & Canters, G.W. Selective observation of the Cu(I)-amicyanin metal site by paramagnetic NMR on partially oxidised samples 1997 J. Biomol. NMR
    Vol. 9(3), pp. 299-305 
    Article DOI
    Citations 
    Abstract: The relaxation enhancement caused by paramagnetic copper(II) is used to observe selectively the metal site of copper(I)-amicyanin by one- and two-dimensional NMR spectroscopy. The paramagnetic effect is communicated to the diamagnetic protein through the electron self-exchange reaction in partially oxidised samples, and call be used for the selective detection of protons around the metal. Relaxation-selective NMR pulse sequences, like super-WEFT and WEFT-NOESY, are used to achieve the desired selection of the signals. The spectra obtained show well-resolved signals corresponding to protons within a radius of similar to 7 Angstrom from the metal, including almost all protons from the coordinated residues. A significant increase in resolution as well as selection of the most relevant part of the protein (close to the active centre) are the principal advantages of this technique, which can be used to obtain specific information about the metal site in blue copper proteins, to assist in the assignment of their NMR spectra and to determine functional properties like the electron self-exchange rate.
    BibTeX:
    @article{Salgado1997,
      author = {J. Salgado and A. P. Kalverda and G. W. Canters},
      title = {Selective observation of the Cu(I)-amicyanin metal site by paramagnetic NMR on partially oxidised samples},
      journal = {J. Biomol. NMR},
      year = {1997},
      volume = {9},
      number = {3},
      pages = {299-305},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1023/A:1018683026421}
    }
    
    Salgado, J., Jimenez, H.R., Moratal, J.M., Kroes, S., Warmerdam, G.C.M. & Canters, G.W. Paramagnetic cobalt and nickel derivatives of Alcaligenes denitrificans azurin and its M121Q mutant. A H-1 NMR study 1996 Biochemistry
    Vol. 35(6), pp. 1810-1819 
    Article DOI
    Citations 
    Abstract: Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic H-1 NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin. Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His46, and His117 metal ligands suggest a movement of the metal ion out of the equatorial plane, indicating that the metal site is tetrahedral. These effects are less pronounced in the Ni(II) M121Q azurin than in the Co(II) metalloderivative. The similarity between the NMR spectra of the Co( II) derivatives of stellacyanin and the M121Q azurin is in agreement with a very similar metal site in both proteins and supports the existence of a coordinated Gin in stellacyanin.
    BibTeX:
    @article{Salgado1996,
      author = {J. Salgado and H. R. Jimenez and J. M. Moratal and S. Kroes and G. C. M. Warmerdam and G. W. Canters},
      title = {Paramagnetic cobalt and nickel derivatives of Alcaligenes denitrificans azurin and its M121Q mutant. A H-1 NMR study},
      journal = {Biochemistry},
      year = {1996},
      volume = {35},
      number = {6},
      pages = {1810-1819},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1021/bi951748a}
    }
    
    Lopez-Camacho, C., Salgado, J., Lequerica, J.L., Madarro, A., Ballestar, E., Franco, L. & Polaina, J. Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A 1996 Biochem. J.
    Vol. 314, pp. 833-838 
    Article URL
    Citations 
    Abstract: Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.
    BibTeX:
    @article{LopezCa1996,
      author = {C. Lopez-Camacho and J. Salgado and J. L. Lequerica and A. Madarro and E. Ballestar and L. Franco and J. Polaina},
      title = {Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A},
      journal = {Biochem. J.},
      year = {1996},
      volume = {314},
      pages = {833-838},
      note = {PT: J; PN: Part 3},
      url = {http://www.biochemj.org/bj/314/0833/bj3140833.htm}
    }
    
    Kroes, S.J., Salgado, J., Parigi, G., Luchinat, C. & Canters, G.W. Electron relaxation and solvent accessibility of the metal site in wild-type and mutated azurins as determined from nuclear magnetic relaxation dispersion experiments 1996 J. Biol. Inorg. Chem.
    Vol. 1(6), pp. 551-559 
    Article DOI
    Citations 
    Abstract: The interaction of water molecules with copper in wild-type azurin and different site-directed mutants of the coordinated residues is studied by nuclear magnetic relaxation dispersion. Different degrees of solvent accessibility are found. The low relaxivity of wild-type azurin agrees with a solvent-protected copper site in solution, the closest water being found at a distance of more than 5 Angstrom from the copper. This low relaxivity contrasts with the relatively large relaxivity of the His46Gly and His117Gly azurin mutants, which shows clear evidence of copper-coordinated water. The data on the latter mutants are best analyzed in terms of one and two water molecules coordinated to the copper in His46Gly and His117Gly, respectively. The Met121His azurin mutant shows an intermediate behavior. The data are analyzed in terms of an increased solvent accessibility with respect to the wild-type azurin, resulting in semi-coordination of water at low pH. These different modes of coordination lead to different geometries, ranging from the trigonal type 1 site of b wild-type azurin to the tetragonal type 2 copper sites of the His117Gly and His46Gly azurin mutants through a so-called type 1.5 site of the Met121His mutant. A correlation is found between the relaxation time (tau(s)) of the unpaired electron of copper(II) and the geometry of the metal site: as the tetragonal character decreases the relaxation becomes significantly faster. tau(s) values of less than or equal to 1 ns are found for the tetrahedrally distorted type 1 and type 1.5 sites and of 5-15 ns for the tetragonal type 2 sites.
    BibTeX:
    @article{Kroes1996,
      author = {S. J. Kroes and J. Salgado and G. Parigi and C. Luchinat and G. W. Canters},
      title = {Electron relaxation and solvent accessibility of the metal site in wild-type and mutated azurins as determined from nuclear magnetic relaxation dispersion experiments},
      journal = {J. Biol. Inorg. Chem.},
      year = {1996},
      volume = {1},
      number = {6},
      pages = {551-559},
      note = {PT: J},
      doi = {href="http://dx.doi.org/10.1007/s007750050091}
    }
    
    Kalverda, A.P., Salgado, J., Dennison, C. & Canters, G.W. Analysis of the paramagnetic copper(II) site of amicyanin by 1H NMR spectroscopy 1996 Biochemistry
    Vol. 35(9), pp. 3085-3092 
    Article DOI
    Citations 
    Abstract: Application of the tailored pulse sequences like super-WEFT allows the direct observation of the hyperfine-shifted signals of the paramagnetic Cu(II) forms of blue copper proteins in solution. The signals can be assigned by applying 2D NMR techniques, like EXSY, to solutions containing a mixture of reduced and oxidized species. The Fermi contact shift is separated from the pseudocontact shift on the basis of the known g-tensor anisotropy of the Cu(II) state, allowing the determination of a number of hyperfine-splitting constants between protons on the Cu ligands and the unpaired electron. These results are used to quantify the spin density distribution over the Cu ligands. In amicyanin about 50%-60% of the unpaired electron density is found on the ligands. It appears possible to quantify the Cu-S(Met) interaction on the basis of the NMR results. Application of the technique to the wild type forms of amicyanin and azurin and to two active site mutants of amicyanin (His96Asp and a plastocyanin-amicyanin loop exchange mutant) shows that the Cu-S(Met) interaction parallels the rhombicity and axial distortion of the Cu site.
    BibTeX:
    @article{Kalverd1996,
      author = {A. P. Kalverda and J. Salgado and C. Dennison and G. W. Canters},
      title = {Analysis of the paramagnetic copper(II) site of amicyanin by 1H NMR spectroscopy},
      journal = {Biochemistry},
      year = {1996},
      volume = {35},
      number = {9},
      pages = {3085-3092},
      note = {LR: 20061115; PUBM: Print; JID: 0370623; 0 (Bacterial Proteins); 0 (Metalloproteins); 0 (Recombinant Proteins); 0 (Solutions); 0 (mauC protein, Methylobacterium extorquens); 12284-43-4 (Azurin); 1333-74-0 (Hydrogen); 7440-50-8 (Copper); EIN: Biochemistry 1996 Aug 13;35(32):10586; ppublish},
      doi = {href="http://dx.doi.org/10.1021/bi9518508}
    }
    
    Jimenez, H.R., Salgado, J., Moratal, J.M. & Morgenstern-Badarau, I. EPR and magnetic susceptibility studies of cobalt(II)- and nickel(II)-substituted azurins from Pseudomonas aeruginosa. Electronic structure of the active sites 1996 Inorg. Chem.
    Vol. 35(10), pp. 2737-2741 
    Article DOI
    Citations 
    Abstract: The electronic properties of cobalt(II)- and nickel(II)-substituted azurins from Pseudomonas aeruginosa have been investigated. EPR data for the cobalt derivative and paramagnetic susceptibility data for the nickel derivative are reported. The EPR spectrum of Co(II)-azurin shows the typical pattern of a Kramers' doublet (+/- 1/2) associated with an S = 3/2 ground state in a distorted axial symmetry environment. The temperature dependence of the EPR intensities shows that this Kramers' doublet is the excited doublet and, therefore, that the corresponding zero-field splitting parameter D is negative (similar to -3.5 cm(-1)). The mean g value is equal to 2.3. Nickel(II) azurin exhibits an effective magnetic moment mu(eff) = 2.8 mu(B) (Bohr magnetons), constant in the temperature range 120-30 K. The magnetic moment decreases and reaches the value of 1.80 mu(B) at 5 K. From the temperature dependence of the susceptibility, the fitting of the data to the theoretical S = 1 susceptibility equation leads to a zero-field splitting parameter D of around 17.7 cm(-1). The spin Hamiltonian parameters that have been determined for the two metallosubstituted proteins are consistent with a highly distorted tetrahedral structure derived from an axially elongated trigonal bipyramid.
    BibTeX:
    @article{Jimenez1996,
      author = {H. R. Jimenez and J. Salgado and J. M. Moratal and I. Morgenstern-Badarau},
      title = {EPR and magnetic susceptibility studies of cobalt(II)- and nickel(II)-substituted azurins from Pseudomonas aeruginosa. Electronic structure of the active sites},
      journal = {Inorg. Chem.},
      year = {1996},
      volume = {35},
      number = {10},
      pages = {2737-2741},
      note = {PT: J}
      doi = {href="http://dx.doi.org/10.1021/ic9513548}
    }
    
    Salgado, J., Jimenez, H.R., Donaire, A. & Moratal, J.M. 1H-NMR study of a cobalt-substituted blue copper protein: Pseudomonas aeruginosa Co(II)-azurin 1995 Eur. J. Biochem.
    Vol. 231(2), pp. 358-369 
    Article DOI
    Citations 
    Abstract: Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1H-NMR spectrum of Pseudomonas aeruginosa Co(II)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact-shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(II)-azurin, i.e. His46, His117, Cys112, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(II) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N2S plane towards the Gly45 O axial position and weakly interacting with the Met121 sulfur.
    BibTeX:
    @article{Salgado1995,
      author = {J. Salgado and H. R. Jimenez and A. Donaire and J. M. Moratal},
      title = {1H-NMR study of a cobalt-substituted blue copper protein: Pseudomonas aeruginosa Co(II)-azurin},
      journal = {Eur. J. Biochem.},
      year = {1995},
      volume = {231},
      number = {2},
      pages = {358-369},
      note = {LR: 20070723; PUBM: Print; JID: 0107600; 12284-43-4 (Azurin); 7440-48-4 (Cobalt); 7440-50-8 (Copper); 7732-18-5 (Water); 7789-20-0 (Deuterium Oxide); ppublish},
      doi = {http://scholar.google.com/scholar?cites=3459045226789196077}
    }
    
    Salgado, J. Metal sustitution in blue copper proteins: Chemical and structural properties of cobalt and nickel azurins. 1995 PhD Thesis,
    University of Valencia, Burjassot, Spain. 
    PhD Thesis URL 
    Abstract:
    BibTeX:
    @phdthesis{Salgado1995a,
      address = {Spain},
      type = {{PhD}},
      author = {Jesús Salgado},
      title = {Metal sustitution in blue copper proteins},
      School = {University of Valencia},
      language = {English}
      year = {1995},
      url = {http://roderic.uv.es/handle/10550/38153}
    }
    
    Moratal, J.M., Romero, A., Salgado, J., Perales-Alarcon, A. & Jimenez, H.R. The crystal structure of nickel(II)-azurin 1995 Eur. J. Biochem.
    Vol. 228(3), pp. 653-657 
    Article DOI
    Citations 
    Abstract: The nickel(II)-azurin metalloderivative has been crystallized and its structure solved at 0.205-nm resolution by X-ray diffraction. The overall structure is not modified by the metal exchange and the only differences with regard to the native copper(II)-azurin occur in the metal site region. These variations affect principally the axial ligands. Nickel co-ordinates more strongly to the carbonyl oxygen of Gly45 while its distance to the Met121 S4 enlarges up to 0.330 nm. The resulting metal center structure is intermediate between those of the Cu(II) and Zn(II) azurins, and can be described as distorted tetrahedral. However, the existence of contact interaction between Met121 and the nickel ion is still possible as has been shown by paramagnetic 1H-NMR studies in solution.
    BibTeX:
    @article{Moratal1995,
      author = {J. M. Moratal and A. Romero and J. Salgado and A. Perales-Alarcon and H. R. Jimenez},
      title = {The crystal structure of nickel(II)-azurin},
      journal = {Eur. J. Biochem.},
      year = {1995},
      volume = {228},
      number = {3},
      pages = {653-657},
      note = {LR: 20070723; PUBM: Print; JID: 0107600; 12284-43-4 (Azurin); 7440-02-0 (Nickel); ppublish},
      doi = {http://dx.doi.org/10.1111/j.1432-1033.1995.0653m.x}
    }
    
    Donaire, A., Salgado, J., Jimenez, H.R. & Moratal, J.M. Cobalt substituted proteins 1995 In Nuclear Magnetic Resonance of Paramagnetic Macromolecules (series: NATO Science, C: Mathematical and Physical Sciences, vol. 457), la Mar, G. (ed.). Kluwer Academic Publishers, Dordrecht, pp. 213-244. Chapter Citations 
    BibTeX:
    @Chapter{Donaire1995,
      author = {Donaire, A. and Salgado, J. and Jimenez, H. R. and Moratal, J. M.},
      title = {Cobalt substituted proteins},
      booktitle = {Nuclear Magnetic Resonance of Paramagnetic Macromolecules},
      series = {NATO Science Series C: Mathematical and Physical Sciences},
      editor = {la Mar, G.N.},
      publisher = {Kluwer Academic Publishers},
      address = {Dordrecht},
      year = {1995},
      volume = {457},
      pages = {213-244}
    }
    
    Moratal, J.M., Castells, J., Donaire, A., Salgado, J., Jimenez, H.R. & Domingo, R. Interaction of cobalt ions with carboxypeptidase A 1994 J. Inorg. Biochem.
    Vol. 53(1), pp. 1-11 
    Article DOI
    Citations 
    Abstract: The interaction of cobalt(II) with native and cobalt(II)-substituted carboxypeptidase has been investigated, at pH 7.5, by electronic absorption and 1H NMR spectroscopies. The reaction of the cobalt(II) uptake by the metalloenzyme [MCPA] (M = Zn or Co) occurs very slowly and a bimetallic complex, [MCPA(Co)], is formed. On the basis of the 1H NOE experiments, the isotropically shifted proton resonances were assigned as belonging to a coordinated histidine residue. 1H NMR titrations of [ZnCPA(Co)] with zinc(II) show that the zinc ion does not compete with cobalt for binding to the noncatalytic site. The temperature dependence of the isotropic shifts, molar absorbance, and longitudinal relaxation time values are indicative of a five-coordinated geometry for the cobalt ion. The identification of the noncatalytic cobalt binding site is also discussed.
    BibTeX:
    @article{Moratal1994,
      author = {J. M. Moratal and J. Castells and A. Donaire and J. Salgado and H. R. Jimenez and R. Domingo},
      title = {Interaction of cobalt ions with carboxypeptidase A},
      journal = {J. Inorg. Biochem.},
      year = {1994},
      volume = {53},
      number = {1},
      pages = {1-11},
      note = {LR: 20061115; PUBM: Print; JID: 7905788; 7440-48-4 (Cobalt); 7440-66-6 (Zinc); EC 3.4.- (Carboxypeptidases); EC 3.4.17.1 (Carboxypeptidases A); ppublish},
      doi = {http://dx.doi.org/10.1016/0162-0134(94)80016-2}
    }
    
    Moratal, J.M., Salgado, J., Donaire, A., Jimenez, H.R., Castells, J. & Martinezferrer, M.J. 1H 2d-Nmr Characterization of Ni(ii)-Substituted Azurin from Pseudomonas-Aeruginosa 1993 Magn. Reson. Chem.
    Vol. 31, pp. S41-S46 
    Article DOI
    Citations 
    Abstract: H-1 two-dimensional NMR experiments on nickel(II)-substituted azurin have been successfully applied. Despite the short relaxation times of the hyperfine-shifted resonances, the combined use of NOESY and COSY spectra allowed the assignment of 15 resonances belonging to the metal-coordinated residues Gly-45, His-46, His-117 and Met-121. Even in the case of the two broad and furthest downfield resonances, the NOESY spectra were successful in assigning these signals to the beta-CH2 protons of Cys-112 The protons of the non-coordinated residues Met-13, Phe-15 and Trp-48 were also assigned via NOESY, COSY and TOCSY experiments.
    BibTeX:
    @article{Moratal1993d,
      author = {J. M. Moratal and J. Salgado and A. Donaire and H. R. Jimenez and J. Castells and M. J. Martinezferrer},
      title = {H-1 2d-Nmr Characterization of Ni(ii)-Substituted Azurin from Pseudomonas-Aeruginosa},
      journal = {Magn. Reson. Chem.},
      year = {1993},
      volume = {31},
      pages = {S41-S46},
      note = {PT: J; SI: Sp. Iss. SI},
      doi = {http://dx.doi.org/10.1002/mrc.1260311310}
    }
    
    Moratal, J.M., Salgado, J., Donaire, A., Jimenez, H.R. & Castells, J. 1d-Nmr and 2d-Nmr Studies of the Ph Effects on the Metal-Site Geometry in Nickel(ii)-Azurin from Pseudomonas-Aeruginosa 1993 J. Chem. Soc. Chem. Commun.
    (2), pp. 110-112 
    Article DOI
    Citations 
    Abstract: The isotropically shifted proton resonances in nickel(II)-azurin have been assigned on a firm basis by using 1D- and 2D-NMR methods which has allowed the subtle structural changes on the metal binding site associated with the pH induced conformational change in this protein to be revealed.
    BibTeX:
    @article{Moratal1993c,
      author = {J. M. Moratal and J. Salgado and A. Donaire and H. R. Jimenez and J. Castells},
      title = {1d-Nmr and 2d-Nmr Studies of the Ph Effects on the Metal-Site Geometry in Nickel(ii)-Azurin from Pseudomonas-Aeruginosa},
      journal = {J. Chem. Soc. Chem. Commun.},
      year = {1993},
      number = {2},
      pages = {110-112},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1039/C39930000110}
    }
    
    Moratal, J.M., Salgado, J., Donaire, A., Jimenez, H.R. & Castells, J. Cosy and Noesy Characterization of Cobalt(ii)-Substituted Azurin from Pseudomonas-Aeruginosa 1993 Inorg. Chem.
    Vol. 32(17), pp. 3587-3588 
    Article DOI
    Citations 
    BibTeX:
    @article{Moratal1993b,
      author = {J. M. Moratal and J. Salgado and A. Donaire and H. R. Jimenez and J. Castells},
      title = {Cosy and Noesy Characterization of Cobalt(ii)-Substituted Azurin from Pseudomonas-Aeruginosa},
      journal = {Inorg. Chem.},
      year = {1993},
      volume = {32},
      number = {17},
      pages = {3587-3588},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1021/ic00069a006}
    }
    
    Moratal, J.M., Donaire, A., Salgado, J., Jimenez, H.R., Castells, J. & Piccioli, M. Two-dimensional 1H NMR spectra of ferricytochrome c551 from Pseudomonas aeruginosa 1993 FEBS Lett.
    Vol. 324(3), pp. 305-308 
    Article DOI
    Citations 
    Abstract: The full assignment of 1H NMR signals of heme proton resonances of ferricytochrome c551 from Pseudomonas aeruginosa has been performed by means of 2D NMR experiments. This technique allows the complete and unequivocal assignment of all heme resonances, including methylene resonances of the propionic groups, directly implicated in the pH dependence of the redox properties of cytochrome c551.
    BibTeX:
    @article{Moratal1993,
      author = {J. M. Moratal and A. Donaire and J. Salgado and H. R. Jimenez and J. Castells and M. Piccioli},
      title = {Two-dimensional 1H NMR spectra of ferricytochrome c551 from Pseudomonas aeruginosa},
      journal = {FEBS Lett.},
      year = {1993},
      volume = {324},
      number = {3},
      pages = {305-308},
      note = {LR: 20061115; PUBM: Print; JID: 0155157; 0 (Bacterial Proteins); 0 (Cytochrome c Group); 0 (Ferric Compounds); 14875-96-8 (Heme); 9048-77-5 (cytochrome C(551)); ppublish},
      doi = {http://dx.doi.org/10.1016/0014-5793(93)80140-P}
    }
    
    Marquez, F., Quintana, E., Roca, I. & Salgado, J. Physical-Mechanical Effects of Nd-Yag Laser on the Surface of Sound Dental Enamel 1993 Biomaterials
    Vol. 14(4), pp. 313-316 
    Article DOI
    Citations 
    Abstract: Human dental enamel samples were irradiated using a 5 kHz Q-Switched Nd:YAG laser. An increase in Knoop microhardness and modification of the membrane permselectivity were detached. These results and the changes observed by SEM, can be connected with the fusion of the enamel surface.
    BibTeX:
    @article{Marquez1993,
      author = {F. Marquez and E. Quintana and I. Roca and J. Salgado},
      title = {Physical-Mechanical Effects of Nd-Yag Laser on the Surface of Sound Dental Enamel},
      journal = {Biomaterials},
      year = {1993},
      volume = {14},
      number = {4},
      pages = {313-316},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1016/0142-9612(93)90124-K}
    }
    
    Quintana, E., Marquez, F., Roca, I., Torres, V. & Salgado, J. Some Morphological-Changes Induced by Nd-Yag Laser on the Noncoated Enamel Surface - a Scanning Electron-Microscopy Study 1992 Lasers Surg. Med.
    Vol. 12(2), pp. 131-136 
    Article
     
    Abstract: The enamel surface layer of some human teeth was treated with the low-energy Nd:YAG laser at 8 mJ pulse energy. These samples were previously etched with 0.05 M orthophosphoric acid to reduce the surface reflection. The treated samples, as well as the control samples, were widely studied by scanning electron microscopy, and, in the lased group, significant morphologic changes affecting the enamel surface were observed. Those changes reveal principally the loss of the typical surface structure of the acid-etched enamel. The hydroxyapatite prisms were not discernible, and there was a decrease in the roughness of the lased surface enamel. These laser-induced structural changes may be related to the increased resistance to the calcification reported by many authors.
    BibTeX:
    @article{Quintan1992,
      author = {E. Quintana and F. Marquez and I. Roca and V. Torres and J. Salgado},
      title = {Some Morphological-Changes Induced by Nd-Yag Laser on the Noncoated Enamel Surface - a Scanning Electron-Microscopy Study},
      journal = {Lasers Surg. Med.},
      year = {1992},
      volume = {12},
      number = {2},
      pages = {131-136},
      note = {PT: J}
    }
    
    Moratal, J.M., Martinez-Ferrer, M.J., Jimenez, H.R., Donaire, A., Castells, J. & Salgado, J. 1H NMR and UV-Vis spectroscopic characterization of sulfonamide complexes of nickel(II)-carbonic anhydrase. Resonance assignments based on NOE effects 1992 J. Inorg. Biochem.
    Vol. 45(4), pp. 231-243 
    Article DOI
    Citations 
    Abstract: The binding of acetazolamide, p-fluorobenzensulfonamide, p-toluenesulfonamide, and sulfanilamide to nickel(II)-substituted carbonic anhydrase II has been studied by 1H NMR and electronic absorption spectroscopies. These inhibitors bind to the metal ion forming 1:1 complexes and their affinity constants were determined. The 1H NMR spectra of the formed complexes show a number of isotropically shifted signals corresponding to the histidine ligands. The complexes with benzene-sulfonamides gave rise to very similar 1H NMR spectra. The NMR data suggest that these aromatic sulfonamides bind to the metal ion altering its coordination sphere. In addition, from the temperature dependence of 1H NMR spectra of the p-fluorobenzenesulfonamide adduct, a conformational change is suggested. The T1 values of the meta-like protons of the coordinated histidines have been measured and resonance assignments based on NOE experiments were performed.
    BibTeX:
    @article{Moratal1992b,
      author = {J. M. Moratal and M. J. Martinez-Ferrer and H. R. Jimenez and A. Donaire and J. Castells and J. Salgado},
      title = {1H NMR and UV-Vis spectroscopic characterization of sulfonamide complexes of nickel(II)-carbonic anhydrase. Resonance assignments based on NOE effects},
      journal = {J. Inorg. Biochem.},
      year = {1992},
      volume = {45},
      number = {4},
      pages = {231-243},
      note = {LR: 20061115; PUBM: Print; JID: 7905788; 0 (Sulfonamides); 59-66-5 (Acetazolamide); 7440-02-0 (Nickel); EC 4.2.1.1 (Carbonic Anhydrases); ppublish},
      doi = {http://dx.doi.org/10.1016/0162-0134(92)84012-C}
    }
    
    Moratal, J.M., Donaire, A., Castells, J., Jimenez, H.R., Salgado, J. & Hillerns, F. Spectroscopic Studies of the Interaction of Nickel(ii) Carboxypeptidase with Phosphate and Pyrophosphate 1992 J. Chem. Soc. Dalton Trans.
    (4), pp. 713-717 
    Article DOI
    Citations 
    Abstract: The interaction between nickel(II)-substituted carboxypeptidase A and the inhibitors phosphate and pyrophosphate has been investigated at pH 7 by electronic absorption and H-1, P-31, and C-13 NMR spectroscopies. Upon binding to the nickel enzyme, the longitudinal relaxation rates T1(-1) of the P-31 nucleus of these inhibitors are enhanced significantly compared to the diamagnetic zinc enzyme. The Ni(II) ... P-31 distances indicate that both inhibitors bind directly to the metal ion under rapid-exchange conditions. From the temperature dependence of the isotropic shifts and the molar absorbance values of the 1:1 adducts formed, a pseudo-octahedral co-ordination for the metal ion is suggested. Proton NMR titrations of nickel(II) carboxypeptidase with phosphate in the presence and absence of D-phenylalanine (D-Phe) indicate that the occupancy of the S'1 subsite of the enzyme does not substantially modify the affinity of the phosphate anion for the metal ion. Carbon-13 NMR T2(-1) measurements on (CO2)-C-13-labelled D-Phe show that phosphate or pyrophosphate do not compete for binding to Arg-145. These results are discussed in terms of the general model of anion interaction with carboxypeptidase.
    BibTeX:
    @article{Moratal1992a,
      author = {J. M. Moratal and A. Donaire and J. Castells and H. R. Jimenez and J. Salgado and F. Hillerns},
      title = {Spectroscopic Studies of the Interaction of Nickel(ii) Carboxypeptidase with Phosphate and Pyrophosphate},
      journal = {J. Chem. Soc. Dalton Trans.},
      year = {1992},
      number = {4},
      pages = {713-717},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1039/DT9920000713}
    }
    
    Moratal, J.M., Castells, J., Donaire, A., Salgado, J. & Jimenez, H.R. Interaction of Carboxylate Inhibitors with the Active-Site of Nickel(ii) Carboxypeptidase-a 1992 J. Chem. Soc. Dalton Trans.
    (23), pp. 3317-3324 
    Article DOI
    Citations 
    Abstract: The binding of several carboxylate inhibitors to nickel(II)-substituted carboxypeptidase A, NiCPA, has been investigated through H-1, C-13 NMR and electronic absorption spectroscopies. Both beta-phenyl-propionate and phenylacetate interact with NiCPA forming two complexes of stoichiometries 1:1 and 1:2 and their affinity constants were determined. Whereas the first inhibitor molecule binds at a non-metallic site, the second binds directly to the metal ion in slow exchange on the chemical shift time-scale. Proton nuclear Overhauser effect measurements have been performed on the 1 :2 beta-phenylpropionate complex, allowing a full correlation between the isotropically shifted signals. From the H-1 T1 values of the meta-like protons of the co-ordinated histidines and the molar absorbances of the 1:2 complexes formed, five-co-ordination for the nickel ion is suggested. The NMR data indicate that upon binding of the carboxylate to the metal ion a conformational change occurs. In contrast, acetate displays no evidence for more than a single binding mode to the nickel enzyme. Thus H-1 and C-13 NMR data indicate that acetate binds to the metal ion forming a 1:1 complex in a fast-exchange regime. The Ni ... C-13 distance, r = 2.7 angstrom, calculated by means of the Solomon equation, is consistent with direct co-ordination of the acetate to the metal ion.
    BibTeX:
    @article{Moratal1992,
      author = {J. M. Moratal and J. Castells and A. Donaire and J. Salgado and H. R. Jimenez},
      title = {Interaction of Carboxylate Inhibitors with the Active-Site of Nickel(ii) Carboxypeptidase-a},
      journal = {J. Chem. Soc. Dalton Trans.},
      year = {1992},
      number = {23},
      pages = {3317-3324},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1039/DT9920003317}
    }
    
    Bertini, I., Donaire, A., Monnanni, R., Moratal, J.M. & Salgado, J. Spectroscopic Characterization of Nickel(ii) Carboxypeptidase 1992 J. Chem. Soc. Dalton Trans.
    (8), pp. 1443-1447 
    Article DOI
    Citations 
    Abstract: The electronic and H-1 NMR spectra of nickel-substituted carboxypeptidase have been measured and discussed in the light of the available X-ray data. The interaction with D-phenylalanine has been investigated as well as that with azide. It is proposed that D-Phe binds first at a non-metallic site, probably Arg-145, and then to the metal. The behaviour of L-Phe is more complex. Azide binds at the metal in the presence of the amino acid. These results are similar to those obtained for the parent (zinc) and cobalt-substituted enzymes. Implications for the enzymatic mechanism are discussed.
    BibTeX:
    @article{Bertini1992,
      author = {I. Bertini and A. Donaire and R. Monnanni and J. M. Moratal and J. Salgado},
      title = {Spectroscopic Characterization of Nickel(ii) Carboxypeptidase},
      journal = {J. Chem. Soc. Dalton Trans.},
      year = {1992},
      number = {8},
      pages = {1443-1447},
      note = {PT: J},
      doi = {http://dx.doi.org/10.1039/DT9920001443}
    }
    
    Moratal, J.M., Martinez-Ferrer, M.J., Donaire, A., Castells, J., Salgado, J. & Jimenez, H.R. Spectroscopic Studies of Nickel(ii) Carbonic-Anhydrase and its Adducts with Inorganic Anions 1991 J. Chem. Soc. Dalton Trans.
    (12), pp. 3393-3399 
    Article DOI
    Citations 
    Abstract: Nickel(II) carbonic anhydrase, NiBCA II, and its adducts with nitrate, acetate, cyanate and azide, have been investigated through H-1 NMR and electronic absorption spectroscopies. From the pH dependence of the molar absorbance the acidity constants of NiBCA were determined. The anions bind to the metal ion forming 1:1 adducts, and the corresponding affinity constants have been determined. The H-1 NMR spectra of NiBCA and its adducts have been recorded, and the signals corresponding to the meta-like protons of the co-ordinated histidines followed by H-1 NMR titration. The T1 values of these signals were measured and resonance assignments made based on nuclear Overhauser enhancement experiments. The co-ordination geometry of the metal ion in NiBCA and its adducts is discussed on the basis of the temperature dependence of the isotropic shifts, molar absorbance, and longitudinal relaxation times.
    BibTeX:
    @article{Moratal1991a,
      author = {J. M. Moratal and M. J. Martinez-Ferrer and A. Donaire and J. Castells and J. Salgado and H. R. Jimenez},
      title = {Spectroscopic Studies of Nickel(ii) Carbonic-Anhydrase and its Adducts with Inorganic Anions},
      journal = {J. Chem. Soc. Dalton Trans.},
      year = {1991},
      number = {12},
      pages = {3393-3399},
      note = {PT: J},
      doi = {href="http://dx.doi.org/10.1039/DT9910003393}
    }
    
    Moratal, J.M., Donaire, A., Salgado, J., Castells, J., Domingo, R., Ferrer, M.J.M. & Soler, V. Study of the Effect of several Pseudoproducts of the Enzymatic-Hydrolysis on the Binding of Phosphate and Pyrophosphate to the Cobalt(ii)-Peptidase Substituted Carboxy-Peptidase a by Uv-Vis and (Hp)-H-1-P-31 Nmr Spectroscopies 1991 Anales De Quimica
    Vol. 87(6), pp. 718-725 
    Article
     
    Abstract: The interaction of the inhibitors phosphate and pyrophosphate with cobalt(II)-substituted Carboxypeptidase, CoCPA, in presence of C-terminal pseudoproducts of ester or peptide hydrolysis, D-phenylalanine, L-phenylalanine, phenylacetate and beta-phenylpropionate, has been investigated through UV-vis and H-1, P-31 NMR spectroscopies. The affinity constants of phosphate and pyrophosphate for the adducts CoCPA(L) [L = aminoacid or carboxylate] have been determined by UV-vis and H-1 NMR spectroscopies. Upon binding to the cobalt adduct, CoCPA(L), the longitudinal and transverse relaxation rates T1(-1) and T2(-2) of the P-31 nucleus of these inhibitors are enhanced significantly compared to the corresponding zinc adduct. The cobalt...P-31 distances, calculated by means of the Solomon equation, indicate that phosphate and pyrophosphate bind directly to the metal ion. Whereas binding of phosphate causes only a distortion of the chromophore without alteration of the overall pentacoordinated geometry of the cobalt (II) ion, binding of pyrophosphate causes the metal to switch from pentacoordinate to hexacoordinate. Tipically the metal ion binds anionic ligands, with significant affinity, when aromatic aminoacids or carboxylates are bound in the S'1 subsite of Carboxypeptidase. However, the affinity of phosphate and pyrophosphate is only slightly modified when the S'1 subsite of the enzyme is occupied by pseudoproducts of ester or peptide hydrolysis.
    BibTeX:
    @article{Moratal1991,
      author = {J. M. Moratal and A. Donaire and J. Salgado and J. Castells and R. Domingo and M. J. M. Ferrer and V. Soler},
      title = {Study of the Effect of several Pseudoproducts of the Enzymatic-Hydrolysis on the Binding of Phosphate and Pyrophosphate to the Cobalt(ii)-Peptidase Substituted Carboxy-Peptidase a by Uv-Vis and (Hp)-H-1-P-31 Nmr Spectroscopies},
      journal = {Anales De Quimica},
      year = {1991},
      volume = {87},
      number = {6},
      pages = {718-725},
      note = {PT: J}
    }
    
    Moratal, J.M., Donaire, A., Salgado, J. & Martinez-Ferrer, M.J. Interaction of sulphate and chloride with cobalt(II)-carbonic anhydrase 1990 J. Inorg. Biochem.
    Vol. 40(3), pp. 245-253 
    Article DOI
    Citations 
    Abstract: The interaction between Cobalt(II)-Bovine Carbonic Anhydrase II and the inhibitors sulphate and chloride have been investigated through 1H NMR and electronic absorption spectroscopies. Both inhibitors bind to the metal ion forming a 1:1 adduct and the corresponding affinity constants have been determined. These inhibitors interact weakly with CoBCA II and this interaction only occurs at low pH values. The T1 values of the meta-like protons of the coordinated histidines have been measured. The coordination number of the metal ion in the adducts is discussed on the basis of temperature dependence of the isotropic shifts, T1, and molar absorbance values.
    BibTeX:
    @article{Moratal1990,
      author = {J. M. Moratal and A. Donaire and J. Salgado and M. J. Martinez-Ferrer},
      title = {Interaction of sulphate and chloride with cobalt(II)-carbonic anhydrase},
      journal = {J. Inorg. Biochem.},
      year = {1990},
      volume = {40},
      number = {3},
      pages = {245-253},
      note = {LR: 20061115; PUBM: Print; JID: 7905788; 0 (Carbonic Anhydrase Inhibitors); 0 (Chlorides); 0 (Sulfates); 7440-48-4 (Cobalt); EC 4.2.1.1 (Carbonic Anhydrases); ppublish},
      doi = {href="http://dx.doi.org/10.1016/0162-0134(90)80058-6}
    }
    

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