Biomedical subjects

Jorge A Vila

Publications and source records attributed to Jorge A Vila.

13 recordsLinked to original sources

PubMed2006

Effects of side-chain orientation on the 13C chemical shifts of antiparallel beta-sheet model peptides.

The dependence of the (13)C chemical shift on side-chain orientation was investigated at the density functional level for a two-strand antiparallel beta-sheet model peptide represented by the amino acid sequence Ac-(Ala)(3)-X-(Ala)(12)-NH(2) where X represents any of the 17 naturally occurring amino acids, i.e., not including alanine, glycine and proline. The dihedral angles adopted for the backbone were taken from, and fixed at, observed experimental values of an antiparallel beta-sheet. We carried out a cluster analysis of the ensembles of conformations generated by considering the side-chain dihedral angles for each residue X as variables, and use them to compute the (13)C chemical shifts at the density functional theory level. It is shown that the adoption of the locally-dense basis set approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 17 naturally occurring amino acids in an antiparallel beta-sheet, there is (i) good agreement between computed and observed (13)C(alpha) and (13)C(beta) chemical shifts, with correlation coefficients of 0.95 and 0.99, respectively; (ii) significant variability of the computed (13)C(alpha) and (13)C(beta) chemical shifts as a function of chi(1) for all amino acid residues except Ser; and (iii) a smaller, although significant, dependence of the computed (13)C(alpha) chemical shifts on chi(xi) (with xi > or = 2) compared to chi(1) for eleven out of seventeen residues. Our results suggest that predicted (13)C(alpha) and (13)C(beta) chemical shifts, based only on backbone (phi,psi) dihedral angles from high-resolution X-ray structure data or from NMR-derived models, may differ significantly from those observed in solution if the dihedral-angle preferences for the side chains are not taken into account.

Amino Acid Sequence↗

PubMed2006

Polyproline II conformation is one of many local conformational states and is not an overall conformation of unfolded peptides and proteins.

The alanine-based peptide Ac-XX(A)7OO-NH2, referred to as XAO (where X, A, and O denote diaminobutyric acid, alanine, and ornithine, respectively), has recently been proposed to possess a well defined polyproline II (P(II)) conformation at low temperatures. Based on the results of extensive NMR and CD investigations combined with theoretical calculations, reported here, we present evidence that, on the contrary, this peptide does not have any significant amount of organized P(II) structure but exists in an ensemble of conformations with a distorted bend in the N- and C-terminal regions. The conformational ensemble was obtained by molecular dynamics/simulated annealing calculations using the amber suite of programs with time-averaged distance and dihedral-angle restraints obtained from rotating-frame nuclear Overhauser effect (ROE) volumes and vicinal coupling constants 3J(HN Eta alpha), respectively. The computed ensemble-averaged radius of gyration Rg (7.4 +/- 1.0) A is in excellent agreement with that measured by small-angle x-ray scattering (SAXS) whereas, if the XAO peptide were in the P(II) conformation, Rg would be 11.6 A. Depending on the pH, peptide concentration, and temperature, the CD spectra of XAO do or do not possess the maximum with positive ellipticity in the 217-nm region, which is characteristic of the P(II) structure, reflecting a shifting conformational equilibrium rather than an all-or-none transition. The "P(II) conformation" should, therefore, be considered as one of the accessible conformational states of individual amino acid residues in peptides and proteins rather than as a structure of most of the chain in the early stage of folding.

Alanine↗

PubMed2005

Coupling between conformation and proton binding in proteins.

Interest centers here on whether the use of a fixed charge distribution of a protein solute, or a treatment that considers proton-binding equilibria by solving the Poisson equation, is a better approach to discriminate native from non-native conformations of proteins. In this analysis of the charge distribution of 7 proteins, we estimate the solvation free energy contribution to the total free energy by exploring the 2(zeta) possible ionization states of the whole molecule, with zeta being the number of ionizable groups in the amino acid sequence, for every conformation in the ensembles of 7 proteins. As an additional consideration of the role of electrostatic interactions in determining the charge distribution of native folds, we carried out a comparison of alternative charge assignment models for the ionizable residues in a set of 21 native-like proteins. The results of this work indicate that (1) for 6 out of 7 proteins, estimation of solvent polarization based on the Generalized Born model with a fixed charge distribution provides the optimal trade-off between accuracy, with respect to the Poisson equation, and speed when compared to the accessible surface area model; for the seventh protein, consideration of all possible ionization states of the whole molecule appears to be crucial to discriminate the native from non-native conformations; (2) significant differences in the degree of ionization and hence the charge distribution for native folds are found between the different charge models examined; (3) the stability of the native state is determined by a delicate balance of all the energy components, and (4) conformational entropy, and hence the dynamics of folding, may play a crucial role for a successful ab initio protein folding prediction.

Ions↗

PubMed2005

On the orientation of the backbone dipoles in native folds.

The role of electrostatic interactions in determining the native fold of proteins has been investigated by analyzing the alignment of peptide bond dipole moments with the local electrostatic field generated by the rest of the molecule with and without solvent effects. This alignment was calculated for a set of 112 native proteins by using charges from a gas phase potential. Most of the peptide dipoles in this set of proteins are on average aligned with the electrostatic field. The dipole moments associated with alpha-helical conformations show the best alignment with the electrostatic field, followed by residues in beta-strand conformations. The dipole moments associated with other secondary structure elements are on average better aligned than in randomly generated conformations. The alignment of a dipole with the local electrostatic field depends on both the topology of the native fold and the charge distribution assumed for all of the residues. The influences of (i) solvent effects, (ii) different sets of charges, and (iii) the charge distribution assumed for the whole molecule were examined with a subset of 22 proteins each of which contains <30 ionizable groups. The results show that alternative charge distribution models lead to significant differences among the associated electrostatic fields, whereas the electrostatic field is less sensitive to the particular set of the adopted charges themselves (empirical conformational energy program for peptides or parameters for solvation energy).

Models, Molecular↗

PubMed2005

Interplay of charge distribution and conformation in peptides: comparison of theory and experiment.

We assessed the correlation between charge distribution and conformation of flexible peptides by comparing the theoretically calculated potentiometric-titration curves of two model peptides, Ac-Lys5-NHMe (a model of poly-L-lysine) and Ac-Lys-Ala11-Lys-Gly2-Tyr-NH2 (P1) in water and methanol, with the experimental curves. The calculation procedure consisted of three steps: (i) global conformational search of the peptide under study using the electrostatically driven Monte Carlo (EDMC) method with the empirical conformational energy program for peptides (ECEPP)/3 force field plus the surface-hydration (SRFOPT) or the generalized Born surface area (GBSA) solvation model as well as a molecular dynamics method with the assisted model building and energy refinement (AMBER)99/GBSA force field; (ii) reevaluation of the energy in the pH range considered by using the modified Poisson-Boltzmann approach and taking into account all possible protonation microstates of each conformation, and (iii) calculation of the average degree of protonation of the peptide at a given pH value by Boltzmann averaging over conformations. For Ac-Lys5-NHMe, the computed titration curve agrees qualitatively with the experimental curve of poly-L-lysine in 95% methanol. The experimental titration curves of peptide P1 in water and methanol indicate a remarkable downshift of the first pK(a) value compared to the values for reference compounds (n-butylamine and phenol, respectively), suggesting the presence of a hydrogen bond between the tyrosine hydroxyl oxygen and the H(epsilon) proton of a protonated lysine side chain. The theoretical titration curves agree well with the experimental curves, if conformations with such hydrogen bonds constitute a significant part of the ensemble; otherwise, the theory predicts too small a downward pH shift.

Electrochemistry↗

PubMed2004

Fast and accurate computation of the 13C chemical shifts for an alanine-rich peptide.

The purpose of this work is, first, to present a fast and accurate technique to compute Boltzmann-averaged values of the quantum-chemical 13C chemical shifts for each amino acid in oligopeptides, demonstrated here by an application to the peptide Ac-XXAAAAAAAOO-NH2 (where X denotes diaminobutyric acid, A is alanine, and O is ornithine) [XAO] and, second, to discuss the capability of the 13Calpha and 13Cbeta chemical shifts to distinguish the PP(II) conformation from the alpha-helix and statistical-coil conformations. Use is made of a combination of approaches, summarized as follows: (1) derivation of an ensemble of conformations by using a molecular mechanics technique; (2) use of a clustering procedure to form families and build a reduced set of conformations consisting of the lowest-energy conformations of each family, and (3) computation of the 13C chemical shifts for the lowest-energy conformations of each family, using a quantum-chemical approach that treats a selected residue, or group of residues, with a 6-311+G(2d,p) locally-dense basis set, while the remaining residues in the sequence are treated with a 3-21G basis set. The whole procedure is quite accurate and speeds up the computation of the Boltzmann-averaged values of the 13C-chemical shifts by several orders of magnitude. The present application sheds some light on the conformational preference for alanine and non-alanine residues to occupy the PP(II) helical region of the Ramachandran map.

Alanine↗

PubMed2004

Folding of the villin headpiece subdomain from random structures. Analysis of the charge distribution as a function of pH.

The structure of the 36 residue villin headpiece subdomain is investigated with the electrostatically driven Monte Carlo method. The ECEPP/3 (Empirical Conformational Energy Program for Peptides) force field, plus two different continuum solvation models, were used to describe the conformational energy of the chain with both blocked and unblocked N and C termini. A statistical analysis of an ensemble of ab initio generated conformations was carried out, based on a comparison with a set of ten native-like structures derived from published experimental data, by using rigid geometry and NMR-derived constraints obtained at pH 3.7. The ten native-like structures satisfy the NMR-derived constraints. The whole ensemble of conformations of the terminally unblocked villin headpiece sub-domain, generated by using ECEPP/3 with a continuum solvation model, were subsequently evaluated at pH 3.7 with a potential function that includes ECEPP/3 combined with a fast multigrid boundary element method. At pH 3.7, the lowest-energy conformation found during the conformational search satisfies approximately 70% of both the distance and the dihedral-angle constraints, and possesses the characteristic packing of three phenylalanine residues that constitute the main part of the hydrophobic core of the molecule. On the other hand, computations at pH 3.7 and pH 7.0 for the ten native-like structures satisfying the NMR-derived constraints indicate a substantial change in the charge distribution for each type of amino acid residue with the change in pH. The results of this study provide a basis to understand the effect of the interactions, such as hydrophobicity, charge-charge interaction and solvent polarization, on the stability of this small alpha-helical protein.

Carrier Proteins↗

PubMed2004

Polyproline II helix conformation in a proline-rich environment: a theoretical study.

Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP(II) structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro)(3)-X-(Pro)(3)-Gly-Tyr-NH(2), with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro)(3)-Ala-Ala-(Pro)(3)-Gly-Tyr-NH(2) (PAAP), by using a theoretical approach that includes a solvent effect as well as cis <--> trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since (13)C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles (phi,psi) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged (13)C chemical shifts for the guest residues in the PXP peptide (X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.

Amino Acid Sequence↗

PubMed2004

Position dependence of the 13C chemical shifts of alpha-helical model peptides. Fingerprint of the 20 naturally occurring amino acids.

The position dependence of the (13)C chemical shifts was investigated at the density functional level for alpha-helical model peptides represented by the sequence Ac-(Ala)(i)-X-(Ala)(j)-NH(2), where X represents any of the 20 naturally occurring amino acids, with 0 < or = i < or = 8 and i + j = 8. Adoption of the locally dense basis approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 20 naturally occurring amino acids in alpha-helices, there is (1) significant variability of the computed (13)C shielding as a function of both the guest residue (X) and the position along the sequence; for example, at the N terminus, the (13)C(alpha) and (13)C(beta) shieldings exhibit a uniform pattern of variation with respect to both the central or the C-terminal positions; (2) good agreement between computed and observed (13)C(alpha) and (13)C(beta) chemical shifts in the interior of the helix, with correlation coefficients of 0.98 and 0.99, respectively; for (13)C(alpha) chemical shifts, computed in the middle of the helix, only five residues, namely Asn, Asp, Ser, Thr, and Leu, exhibit chemical shifts beyond the observed standard deviation; and (3) better agreement for four of these residues (Asn, Asp, Ser, and Thr) only for the computed values of the (13)C(alpha) chemical shifts at the N terminus. The results indicate that (13)C(beta), but not (13)C(beta), chemical shifts are sensitive enough to reflect the propensities of some amino acids for specific positions within an alpha-helix, relative to the N and C termini of peptides and proteins.

Amino Acid Sequence↗

PubMed2003

Atomically detailed folding simulation of the B domain of staphylococcal protein A from random structures.

The conformational space of the 10-55 fragment of the B-domain of staphylococcal protein A has been investigated by using the electrostatically driven Monte Carlo (EDMC) method. The ECEPP/3 (empirical conformational energy program for peptides) force-field plus two different continuum solvation models, namely SRFOPT (Solvent Radii Fixed with atomic solvation parameters OPTimized) and OONS (Ooi, Oobatake, Némethy, and Scheraga solvation model), were used to describe the conformational energy of the chain. After an exhaustive search, starting from two different random conformations, three of four runs led to native-like conformations. Boltzmann-averaged root-mean-square deviations (RMSD) for all of the backbone heavy atoms with respect to the native structure of 3.35 A and 4.54 A were obtained with SRFOPT and OONS, respectively. These results show that the protein-folding problem can be solved at the atomic detail level by an ab initio procedure, starting from random conformations, with no knowledge except the amino acid sequence. To our knowledge, the results reported here correspond to the largest protein ever folded from a random conformation by an initial-value formulation with a full atomic potential, without resort to knowledge-based information.

Hydrogen Bonding↗

PubMed2003

Unblocked statistical-coil tetrapeptides in aqueous solution: quantum-chemical computation of the carbon-13 NMR chemical shifts.

We recently reported a theoretical characterization of representative ensembles of statistical-coil conformations for tetrapeptides with unblocked termini in aqueous solution, at pH 7. The results showed good agreement between the computed Boltzmann-averaged and experimentally-determined values for both the vicinal coupling constants (3)J(NHalpha) and the alpha-proton chemical shifts. Here, we carry out a cluster analysis of the ensembles of conformations generated in that study, and use them to compute the Boltzmann-averaged values of the quantum-chemical (13)C chemical shifts for different amino acids in the unblocked tetrapeptides GGXA (where X stands for Phe, Arg, His, Glu, Ile, Lys, Gln, Tyr, Leu, Thr, Ala, Gly and Val). The values of the (13)C chemical shifts in these thirteen amino acids (for which experimental data are available) were computed by using Density Functional Theory with a 6-311+G(2d,p) basis set. Good agreement is found in terms of both the correlation coefficient (R) and standard deviations of the difference between the computed Bolztmann-averaged and the NMR-determined values for the (13)C chemical shifts. These results suggest that it may be possible to build a reliable theoretically-derived database of chemical shifts for statistical-coil residues. The results of the current study contribute to our understanding of the relations between chemical shifts, dihedral angles and vicinal coupling constants, (3)J(NHalpha). In addition, they can shed light as to how the statistical-coil conformation is related to the conformational preference of more structured states, such as the alpha-helical conformation.

Amino Acid Sequence↗

PubMed2002

Helix-coil transitions re-visited.

The thermally-induced helix-coil transition in polyamino acids is a good model for determining the helix-forming propensities of amino acids but not for the two-state folding/unfolding transition in globular proteins. The equilibrium and kinetic treatments of the helix-coil transition are summarized here together with a description of applications to various types of homopolymers and copolymers. Attention is then focused on the helix-coil transition in poly-L-alanine as an example of a non-polar polyamino acid. To render such a non-polar polymer water soluble, it is necessary to introduce polar amino acids such as lysines, but care must be taken as to the location of such polar residues. If they are attached as end groups, as in a triblock copolymer, they do not perturb the helix-forming tendency of the central poly-L-alanine block significantly, but if they are introduced within the sequence of alanine residues, then the hydration properties of the lysines dominate the behavior of the resulting copolymer, thereby leading to erroneous values of the parameters characterizing the helix-forming tendency of the alanines. Neutral but polar residues, such as glutamines, also exhibit hydration-dominating properties but less so than charged lysines. Some details of the calculations for an alanine/glutamine copolymer are presented here. It is concluded that random copolymers based on a neutral water-soluble host provide reliable information about the helix-forming tendencies of amino acid residues that are introduced as guests among such neutral host residues.

Amino Acid Sequence↗

PubMed2002

Unblocked statistical-coil tetrapeptides and pentapeptides in aqueous solution: a theoretical study.

NMR studies of the molecular conformations of peptides and proteins rely on a comparison of the relevant spectral parameters with the corresponding values for so-called statistical-coil polypeptides. For this reason, it is necessary to characterize the experimental ensemble of states populated by statistical-coil peptides. Such a characterization, however, has proven to be both difficult and sensitive to changes in many environmental parameters such as solvent composition, temperature, pH, as well as the neighboring amino acids in the sequence. As a consequence, a series of significant discrepancies has been reported for some experimentally observed parameters, such as chemical shifts, or vicinal coupling constants, (3)J(NHalpha), whose values appear to be incompatible with a statistical-coil ensemble. In this work, we report the results of a molecular mechanics study of a series of unblocked tetra- and pentapeptides under different pH conditions. These calculations were carried out with explicit consideration of both the coupling between the process of proton binding/release and conformation adopted by the molecule at a given pH and the contribution of the conformational entropy to the total free energy. Good agreement was found between the calculated and experimentally determined values of the vicinal coupling constant, (3)J(NHalpha), the alpha-proton chemical shift, and the (13)C(alpha) chemical shift. All the evidence accumulated in these theoretical calculations helps to rationalize some of the unsettled anomalies observed experimentally, and to provide an understanding of the effect of pH and amino acid sequence on the conformational preferences of statistical-coil peptides.

Amino Acids↗