Statistical Analysis Of Protein Structures Research Articles

Contribution of buried aspartic acid to the stability of the PDZ2 protein

Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18kJ·mol−1. These findings are discussed in terms of contribution of buried ionizable groups to protein stability.

Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges

It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ˜2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins.

What is a Desirable Statistical Energy Function for Proteins and How Can It Be Obtained?

Can one obtain a physical energy function for proteins from statistical analysis of protein structures? A direct answer to this question is likely "no." Aless demanding question is whether one can produce a statistical energy function that has the desirable features of a physical-based energy function. Such a desirable energy function would be founded on a physical basis with few or no adjustable parameters, reproduce the known physical characters of amino acid residues, be mostly database independent and transferable, and, more importantly, reasonably accurate in various applications. In this review, we show how such a desirable energy function can be obtained via introducing a simple physical-based reference state called DFIRE (Distance-scaled, Finite, Ideal-gas REference state).

Nh3D: A reference dataset of non-homologous protein structures

BackgroundThe statistical analysis of protein structures requires datasets in which structural features can be considered independently distributed, i.e. not related through common ancestry, and that fulfil minimal requirements regarding the experimental quality of the structures it contains. However, non-redundant datasets based on sequence similarity invariably contain distantly related homologues. Here we provide a reference dataset of non-homologous protein domains, assuming that structural dissimilarity at the topology level is incompatible with recognizable common ancestry. The dataset is based on domains at the Topology level of the CATH database which hierarchically classifies all protein structures. It contains the best refined representatives of each Topology level, validates structural dissimilarity and removes internally duplicated fragments. The compilation of Nh3D is fully scripted.ResultsThe current Nh3D list contains 570 domains with a total of 90780 residues. It covers more than 70% of folds at the Topology level of the CATH database and represents more than 90% of the structures in the PDB that have been classified by CATH. We observe that even though all protein pairs are structurally dissimilar, some pairwise sequence identities after global alignment are greater than 30%.ConclusionNh3D is freely available as a reference dataset for the statistical analysis of sequence and structure features of proteins in the PDB. Regularly updated versions of Nh3D and the corresponding PDB-formatted coordinate sets are accessible from our Web site .

Hydrophobic interactions at the Ccap position of the C-capping motif of alpha-helices.

We investigated the possible role of residues at the Ccap position in an alpha-helix on protein stability. A set of 431 protein alpha-helices containing a C'-Gly from the Protein Data Bank (PDB) was analyzed, and the normalized frequencies for finding particular residues at the Ccap position, the average fraction of buried surface area, and the hydrogen bonding patterns of the Ccap residue side-chain were calculated. We found that on average the Ccap position is 70% buried and noted a significant correlation (R=0.8) between the relative burial of this residue and its hydrophobicity as defined by the Gibbs energy of transfer from octanol or cyclohexane to water. Ccap residues with polar side-chains are commonly involved in hydrogen bonding. The hydrogen bonding pattern is such that, the longer side-chains of Glu, Gln, Arg, Lys, His form hydrogen bonds with residues distal (>+/-4) in sequence, while the shorter side-chains of Asp, Asn, Ser, Thr exhibit hydrogen bonds with residues close in sequence (<+/-4), mainly involving backbone atoms. Experimentally we determined the thermodynamic propensities of residues at the Ccap position using the protein ubiquitin as a model system. We observed a large variation in the stability of the ubiquitin variants depending on the nature of the Ccap residue. Furthermore, the measured changes in stability of the ubiquitin variants correlate with the hydrophobicity of the Ccap residue. The experimental results, together with the statistical analysis of protein structures from the PDB, indicate that the key hydrophobic capping interactions between a helical residue (C3 or C4) and a residue outside the helix (C", C3' or C4') are frequently enhanced by the hydrophobic interactions with Ccap residues.

Effects of amino acid phi,psi propensities and secondary structure interactions in modulating H alpha chemical shifts in peptide and protein beta-sheet.

H alpha chemical shifts are often used as indicators of secondary structure formation in protein structural analysis and peptide folding studies. On the basis of NMR analysis of model beta-sheet and alpha-helical peptides, together with a statistical analysis of protein structures for which NMR data are available, we show that although the gross pattern of H alpha chemical shifts reflects backbone torsion angles, longer range effects from distant amino acids are the dominant factor determining experimental chemical shifts in beta-sheets of peptides and proteins. These show context-dependent variations that aid structural assignment and highlight anomalous shifts that may be of structural significance and provide insights into beta-sheet stability.

Main-chain complementarity in protein-protein recognition.

The existing theoretical approaches to protein-protein recognition concentrate on the details of the molecular surface at atomic resolution, while a possible role of the main chain in complex formation has been largely unexplored. To address this problem, we represented the molecules by C alpha atoms and applied the step-function potentials for intermolecular energy calculations. Since our goal was not to predict, as accurately as possible, the structure of a protein-ligand complex, but to reveal the role of the backbone in the formation of such a complex, all the potentials were identical and C alpha centered. Thus, for the specific purposes of our study, we do not simulate the difference in the side chains at the molecular surface. The structures were taken from known co-crystallized complexes. The intermolecular energy calculation was performed by a systematic 6-D search on a grid. The results revealed that in all cases tested (except antigen-antibody) the positions of the ligand at the binding site on the receptor corresponded to the lowest-energy configurations of the complex. The complementarity between the backbones, in general, may facilitate the initial placement of the ligand at the binding site of the receptor. At the same time, the identity and the specific conformation of the surface side chains play a crucial role in the next stage of the complex formation.

Stabilization of an isolated helical capping box in solution by hydrophobic interactions: evidence from the NMR study of bioactive peptides from the C-terminus of human C5a anaphylatoxin.

Synthetic analogues of the C-terminal portion of C5a were designed and found to be agonists of the C5a receptor [J. A. Ember et al. (1992) Jounral of Immunology, Vol. 148, p. 3165]. Nuclear magnetic resonance experiments were carried out to determine the solution conformation of the most potent analogue, the peptide C5a 65-74 (Tyr65, Phe67) (Tyr65-Ser66-Phe67-Lys68-Asp69-Met70 -Gln71- Leu72-Gly73-Arg74). Medium-range nuclear Overhauser effects (NOEs) were observed for residues 65-70 of this C5a peptide, suggesting that this region adopts a folded conformation in a significant population of the solution conformational ensemble. Quantitative analyses of (3)J(NH-alphaH) coupling constants and sequential NOE cross peaks gave an estimated helical population of 65% in the region Ser66-Met70. Additional evidence supporting the presence of a helical turn includes reduced amide-proton temperature coefficients and lowered (3)J(NH-alphaH) coupling constants in the region of Phe67-Met70. Conformational behavior of this C5a analogue peptide was studied using molecular modeling incorporating observed NOEs as constraints. The side chains of Tyr65, Phe67, and Met70 consistently form a hydrophobic cluster in all the model structures. The side chains of residues Ser66 and Asp69 can form reciprocal hydrogen bonds with the backbone NH groups of these two residues, indicating that residues Ser66-Phe67-Lys68-Asp69 (or SFKD) form a helix-stabilizing capping box (E. T. Harper and G. D. Rose (1993) Biochemistry, Vol. 32, p. 7605; H. X. Zhou et al. (1994) Proteins: Structure, Function and Genetics, Vol. 18, p. 1] even within the single turn of helical structure found in the analogue C5a peptide. A comparison of nmr results obtained for the analogue peptide and the natural decapeptide C5a 65-74 (Ile65-Ser66-His67-Lys68-Asp-69- Met70-Gln71-Leu72-Gly73-Arg74) indicated that incorporation of residues Tyr65 and Phe67 helps stabilize an isolated capping box involving residues Ser66-Asp69 in the C5a peptides through more extensive hydrophobic/aromatic interactions between residues Tyr65, Phe67, and Met70 in the analogue peptide C5a 65-74 (Tyr65, Phe67). These results constitute the first experimental demonstration of hydrophobic determinants in helical capping-box interactions, proposed recently by a statistical analysis of protein structures [J. W. Seale et al. (1994) Protein Science, Vol. 3, pp. 1741-1745]. The stabilized helical turn may also account for the greater potency of the analogue peptide C5a65-74 (Tyr65, Phe67) in receptor-binding assays.

Distance distributions in proteins: a six-parameter representation.

We present a statistical analysis of protein structures based on interatomic C alpha distances. The overall distance distributions reflect in detail the contents of sequence-specific substructures maintained by local interactions (such as alpha-helixes) and longer range interactions (such as disulfide bridges and beta-sheets). We also show that a volume scaling of the distances makes distance distributions for protein chains of different length superimposable. Distance distributions were also calculated specifically for amino acids separated by a given number of residues. Specific features in these distributions are visible for sequence separations of up to 20 amino acid residues. A simple representation, which preserves most of the information in the distance distributions, was obtained using six parameters only. The parameters give rise to canonical distance intervals and when predicting coarse-grained distance constraints by methods such as data-driven artificial neural networks, these should preferably be selected from these intervals. We discuss the use of the six parameters for determining or reconstructing 3-D protein structures.

Thermodynamic β -sheet propensities measured using a zinc-finger host peptide

The three-dimensional structures of proteins reveal that the distribution of amino acids within the major classes of secondary structure is not random but that each amino acid has its own preferred secondary structural arrangements. Propensity scales for residues in alpha-helices have been generated through the use of various host-guest systems. Here we measure the thermodynamic beta-sheet propensities of each of the twenty commonly occurring amino acids. A previously studied zinc-finger peptide was used as the host system in which amino acids were substituted into a guest site, a solvent-exposed position in an antiparallel beta-sheet. As these peptides are unfolded in the absence of bound metal but are folded in their presence, it is assumed that the thermodynamics of metal binding fully reflect peptide-folding energy. A competitive cobalt(II)-binding assay was used to determine these energies with high precision. The relative free energies correlate well with previously derived potential values based on statistical analysis of protein structures. We are therefore able to present a thermodynamic beta-sheet propensity scale for all the commonly occurring amino acids in aqueous solution.

Aromatic-aromatic interactions in the zinc finger motif: Analysis of the two-dimensional nuclear magnetic resonance structure of a mutant domain

The folding and stability of globular proteins are determined by a variety of chemical mechanisms, including hydrogen bonds, salt bridges and the hydrophobic effect. Of particular interest are weakly polar interactions involving aromatic rings, which are proposed to regulate the geometry of closely packed protein interiors. Such interactions reflect the electrostatic contribution of π-electrons and, unlike van der Waals' interactions and the hydrophobic effect, may, in principle, introduce a directional force in a protein's hydrophobic core. Although the weakly polar hypothesis is supported by a statistical analysis of protein structures, the general importance of such contributions to protein folding and stability is unclear. Here, we show the presence of alternative aromaticaromatic interactions in the two-dimensional nuclear magnetic resonance structure of a mutant Zn finger. Changes in aromatic packing lead in turn to local and non-local differences between the structures of a wild-type and mutant domain. The results provide insight into the evolution of Zn finger sequences and have implications for understanding how geometric relationships may be chemically encoded in a simple sequence template.