The difference between protein structures obtained by x-ray analysis and nuclear magnetic resonance

We have analyzed the pairs of protein structures obtained by X-ray diffraction analysis and nuclear magnetic resonance (X-ray and NMR structures) that display no major differences when superimposed on one another (61 pairs). Analyzing atom-to-atom contacts (contact distances 2–8 Å), it has been found that the NMR structures (compared to the X-ray structures) have more contacts at distances below 3.5 Å and above 5.5 Å. In the case of residue-to-residue contacts, the NMR structures have more contacts at distances below 3 Å and between 4.5 and 6.5 Å. At other distances analyzed, the X-ray structures have more contacts. The difference in the numbers of atom-to-atom and residue-to-residue contacts is greater for buried residues inaccessible to water than for surface residues. Another important difference is related to the number of hydrogen bonds in the main chain: this number is greater in the X-ray structures. The coefficient of correlation between the numbers of hydrogen bonds identified in the structures obtained by both methods is only 32%. If a complete set of NMR models of protein structure is considered, the total number of hydrogen bonds proves to be 1.2 times greater than in the X-ray structures, whereas the correlation coefficient increases to only 65%. We have also demon-strated that -helices in the NMR structures are more distorted (compared to the ideal -helix) than those in the X-ray structures.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 129–138.Original Russian Text Copyright © 2005 by Melnik, Garbuzynskiy, Lobanov, Galzitskaya.     

Comparison of X‐ray and NMR structures: Is there a systematic difference in residue contacts between X‐ray‐ and NMR‐resolved protein structures?

We have compared structures of 78 proteins determined by both NMR and X-ray methods. It is shown that X-ray and NMR structures of the same protein have more differences than various X-ray structures obtained for the protein, and even more than various NMR structures of the protein. X-ray and NMR structures of 18 of these 78 proteins have obvious large-scale structural differences that seem to reflect a difference of crystal and solution structures. The other 60 pairs of structures have only small-scale differences comparable with differences between various X-ray or various NMR structures of a protein; we have analyzed these structures more attentively. One of the main differences between NMR and X-ray structures concerns the number of contacts per residue: (1) NMR structures presented in PDB have more contacts than X-ray structures at distances below 3.0 A and 4.5-6.5 A, and fewer contacts at distances of 3.0-4.5 A and 6.5-8.0 A; (2) this difference in the number of contacts is greater for internal residues than for external ones, and it is larger for beta-containing proteins than for all-alpha proteins. Another significant difference is that the main-chain hydrogen bonds identified in X-ray and NMR structures often differ. Their correlation is 69% only. However, analogous difference is found for refined and rerefined NMR structures, allowing us to suggest that the observed difference in interresidue contacts of X-ray and NMR structures of the same proteins is due mainly to a difference in mathematical treatment of experimental results.     

Crystallographic and NMR spectroscopic protein structures: Interresidue contacts

Interresidue pair contacts were analyzed in detail for four pairs of protein structures solved using X-ray analysis (X-ray) and nuclear magnetic resonance (NMR). In the four NMR structures, at distances of ≤4.0 Å, the total number of pair contacts was 4–9% lower and, in general, the pair contacts were 0.02–0.16 Å shorter compared to the X-ray structures. Each of the four structural pairs contained 83–94% common pair contacts (CPCs), which were formed by identical residues in both structures; the other 6–17% were longer intrinsic pair contacts (IPCs) formed by different residues in NMR and X-ray structures, while the latter contained more IPC. Every NMR structure contained three types of CPC that were shorter, longer, or equal to the identical contact pairs in the X-ray structure of this protein. Methodologically different short CPCs prevailed at a known distance dependence of the interresidue contact density in 60–61 pairs of NMR/X-ray structures. Among the analyzed four structural pairs, contact shortening appeared upon the energy minimization of the crambin NMR structure and upon solving the ubiquitin, hen lysozyme, and monomeric hemoglobin NMR structures using X-PLOR software with decreased van der Waals atomic radii. The degree of contact shortening in the NMR structures diminished with an increase in the NMR data used to solve these structures. Among the 60 pairs of NMR/X-ray structures, the major difference between α-helical and β-structural proteins in the dependences on interresidue distances of average contact density appeared due to strong α/β differences in the backbone local geometry.     

Crystallographic and NMR spectroscopic protein structures: the inter-residue contacts

Inter-residue pair contacts have been analyzed in detail for the four pairs of protein structures determined both by X-ray analysis (X-ray) and nuclear magnetic resonance (NMR). At contact distances < or = 4.0 angstroms in the four NMR structures the overall number of pair contacts are less by 4-9% and pair contacts are in average shorter by 0.02-0.16 angstroms than those in corresponding X-ray structures. In each of four structure pairs 83-94% of common pair contacts are formed by the same residues in both structures and rest 6-17% ones are longer own pair contacts formed by the different residues in the NMR and X-ray structures. The amount of the longer own contacts is higher in the X-ray structure of the pair. In the each NMR structure there are three types of common pair contacts, which are shorter, longer or equal length in comparison with identical pair contacts in the X-ray structure of the same protein. The methodological different shortened common pair contacts predominate in the known distant dependence of the inter-residue contact densities of the 60-61 pair of the NMR/X-ray structure. Among four pairs analyzed the contact shortening proceeds upon the energy minimization of the crambin NMR structure and upon the resolving by the program X-PLOR with decreased atom van der Waals radius of the NMR structures of ubiquitin, hen lysozyme and monomeric hemoglobin. An extent of the NMR contact shortening decreased as the amount of NMR information upon the calculation of the NMR structures increased. Among 60-61 pairs of NMR/X-ray structures the main difference between alpha-helical and beta-structural proteins on the inter-residue distant dependence of the average contact densities arises from the strong alpha/beta difference in the local backbone geometry.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.     

Inter-residue and solvent-residue interactions in proteins: a statistical study on experimental structures

A large set of protein structures resolved by X-ray or NMR techniques has been extracted from the Protein Data Bank and analyzed using statistical methods. In particular, we investigate the interactions between side chains and the interactions between solvent and side chains, pointing out on the possibility of including the solvent as part of a knowledge-based potential. The solvent-residue contacts are accounted for on the basis of the Voronoi's polyhedron analysis. Our investigation confirms the importance of hydrophobic residues in determining the protein stability. We observe that in general hydrophobic-hydrophobic interactions and, more specifically, aromatic-aromatic contacts tend to be increasingly distally separated in the primary sequence of proteins, thus connecting distinct secondary structure elements. A simple relation expressing the dependence of the protein free energy by the number of residues is proposed. Such a relation includes both the residue-residue and the solvent-residue contributions. The former is dominant for large size proteins, whereas for small sizes (number of residues less than 100) the two terms are comparable. Gapless threading experiments show that the solvent-residue knowledge-based potential yields a significant contribution with respect to discriminating the native structure of proteins. Such contribution is important especially for proteins of small size and is similar to that given by the most favorable residue-residue knowledge-based potential referring to hydrophobic-hydrophobic interactions such as isoleucine-leucine. In general, the inclusion of the solvent-residue interaction produces a relevant increase of the free energy gap between the native structures and decoys.     

High-precision measurement of hydrogen bond lengths in proteins by nuclear magnetic resonance methods.

We have compared hydrogen bond lengths on enzymes derived with high precision (< or = +/- 0.05 A) from both the proton chemical shifts (delta) and the fractionation factors (phi) of the proton involved with those obtained from protein X-ray crystallography. Hydrogen bond distances derived from proton chemical shifts were obtained from a correlation of 59 O--H....O hydrogen bond lengths, measured by small molecule high-resolution X-ray crystallography, with chemical shifts determined by solid-state nuclear magnetic resonance (NMR) in the same crystals (McDermott A, Ridenour CF, Encyclopedia of NMR, Sussex, U.K.: Wiley, 1996:3820-3825). Hydrogen bond distances were independently obtained from fractionation factors that yield distances between the two proton wells in quartic double minimum potential functions (Kreevoy MM, Liang TM, J Am Chem Soc, 1980;102:3315-3322). The high-precision hydrogen bond distances derived from their corresponding NMR-measured proton chemical shifts and fractionation factors agree well with each other and with those reported in protein X-ray structures within the larger errors (+/-0.2-0.8 A) in distances obtained by protein X-ray crystallography. The increased precision in measurements of hydrogen bond lengths by NMR has provided insight into the contributions of short, strong hydrogen bonds to catalysis for several enzymatic reactions.     

Ribonuclease A. Analysis of the hydrogen bond geometry, and spatial accessibility at the active site

The hydrogen bonding of bovine ribonuclease A derived from the high resolution X-ray structure has been studied in detail. Correlations have been examined for main-chain-main-chain hydrogen bond angles, torsion angles and distances, respectively. Differences are found consistently for correlations associated with alpha-helix and beta-sheet, respectively. Ten of the 124 side-chains have four or more hydrogen bond contacts; two, including Glu-101, have five or more. Three potential C = O---H, three N---X and three potential side-chain H-bonds fail to form. A search for highly inaccessible buried residues resulted in nine outstanding examples, all of which are conserved across 38 known mammalian ribonuclease A sequences, indicating the importance of these residues for structural stability. Of the two histidines in the active site, His-12 has five hydrogen bonds and His-119 three. The conformational space accessible to these two catalytically important residues studied by means of simple non-bonded contact energy calculations confirms the existence of two alternative, interchangeable locations for His-119, while His-12 is locked in a local energy minimum.     

Structure of crambin in solution, crystal and in the trajectories of molecular dynamics simulations

The mechanisms of the three-dimensional crambin structure alterations in the crystalline environments and in the trajectories of the molecular dynamics simulations in the vacuum and crystal surroundings have been analyzed. In the crystalline state and in the solution the partial regrouping of remote intramolecular packing contacts, involved in the formation and stabilization of the tertiary structure of the crambin molecule, occurs in NMR structures. In the crystalline state it is initiated by the formation of the intermolecular contacts, the conformational influence of its appearance is distributed over the structure. The changes of the conformations and positions of the residues of the loop segments, where the intermolecular contacts of the crystal surroundings are preferably concentrated, are most observable. Under the influence of these contacts the principal change of the regular secondary structure of crambin is taking place: extension of the two-strand β structure to the three-strand structure with the participation of the single last residue N46 of the C-terminal loop. In comparison with the C-terminal loop the more profound changes are observed in the conformation and the atomic positions of the backbone atoms and in the solvent accessibility of the residues of the interhelical loop. In the solution of the ensemble of the 8 NMR structures relative accessibility to the solvent differs more noticeably also in the region of the loop segments and rather markedly in the interhelical loop. In the crambin cryogenic crystal structures the positions of the atoms of the backbone and/or side chain of 14–18 of 46 residues are discretely disordered. The disorganizations of at least 8 of 14 residues occur directly in the regions of the intermolecular contacts and another 5 residues are disordered indirectly through the intramolecular contacts with the residues of the intermolecular contacts. Upon the molecular dynamics simulation in the vacuum surrounding as in the solution of the crystalline structure of crambin the essential changes of the backbone conformation are caused by the intermolecular contacts absence, but partly masked by the structure changes owing to the nonpolar H atoms absence on the simulated structure. The intermolecular contact absence is partly manifested upon the molecular dynamics simulation of the crambin crystal with one protein molecule. Compared to the crystal structure the lengths of the interpeptide hydrogen bonds and other interresidue contacts in an average solution NMR structure are somewhat shorter and accordingly the energy of the interpeptide hydrogen bonds is better. This length shortening can occur at the stage of the refinement of the NMR structures of the crambin and other proteins by its energy minimizations in the vacuum surroundings and not exist in the solution protein structures.     

Protein-Protein Docking with Dynamic Residue Protonation States

Protein-protein interactions depend on a host of environmental factors. Local pH conditions influence the interactions through the protonation states of the ionizable residues that can change upon binding. In this work, we present a pH-sensitive docking approach, pHDock, that can sample side-chain protonation states of five ionizable residues (Asp, Glu, His, Tyr, Lys) on-the-fly during the docking simulation. pHDock produces successful local docking funnels in approximately half (79/161) the protein complexes, including 19 cases where standard RosettaDock fails. pHDock also performs better than the two control cases comprising docking at pH 7.0 or using fixed, predetermined protonation states. On average, the top-ranked pHDock structures have lower interface RMSDs and recover more native interface residue-residue contacts and hydrogen bonds compared to RosettaDock. Addition of backbone flexibility using a computationally-generated conformational ensemble further improves native contact and hydrogen bond recovery in the top-ranked structures. Although pHDock is designed to improve docking, it also successfully predicts a large pH-dependent binding affinity change in the Fc–FcRn complex, suggesting that it can be exploited to improve affinity predictions. The approaches in the study contribute to the goal of structural simulations of whole-cell protein-protein interactions including all the environmental factors, and they can be further expanded for pH-sensitive protein design.     

Protein contacts, inter-residue interactions and side-chain modelling

Three-dimensional structures of proteins are the support of their biological functions. Their folds are stabilized by contacts between residues. Inner protein contacts are generally described through direct atomic contacts, i.e. interactions between side-chain atoms, while contact prediction methods mainly used inter-Calpha distances. In this paper, we have analyzed the protein contacts on a recent high quality non-redundant databank using different criteria. First, we have studied the average number of contacts depending on the distance threshold to define a contact. Preferential contacts between types of amino acids have been highlighted. Detailed analyses have been done concerning the proximity of contacts in the sequence, the size of the proteins and fold classes. The strongest differences have been extracted, highlighting important residues. Then, we studied the influence of five different side-chain conformation prediction methods (SCWRL, IRECS, SCAP, SCATD and SCCOMP) on the distribution of contacts. The prediction rates of these different methods are quite similar. However, using a distance criterion between side chains, the results are quite different, e.g. SCAP predicts 50% more contacts than observed, unlike other methods that predict fewer contacts than observed. Contacts deduced are quite distinct from one method to another with at most 75% contacts in common. Moreover, distributions of amino acid preferential contacts present unexpected behaviours distinct from previously observed in the X-ray structures, especially at the surface of proteins. For instance, the interactions involving Tryptophan greatly decrease.     

The propeller DNA conformation of poly(dA).poly(dT).

Physical properties of the DNA duplex, poly(dA).poly(dT) differ considerably from the alternating copolymer poly(dAT). A number of molecular models have been used to describe these structures obtained from fiber X-ray diffraction data. The recent solutions of single crystal DNA dodecamer structures with segments of oligo-A.oligo-T have revealed the presence of a high propeller twist in the AT regions which is stabilized by the formation of bifurcated (three-center) hydrogen bonds on the floor of the major groove, involving the N6 amino group of adenine hydrogen bonding to two O4 atoms of adjacent thymine residues on the opposite strand. Here we show that it is possible to incorporate the features of the single crystal analysis, specifically high propeller twist, bifurcated hydrogen bonds, and a narrow minor groove, as well as the close interstrand NMR signal between adenine HC2 and ribose HC1' of the opposite strand, into a model that is fully compatible with the diffraction data obtained from poly(dA).poly(dT).     

Use of secondary structural information and C<Emphasis Type="Italic">α</Emphasis>-C<Emphasis Type="Italic">α</Emphasis> distance restraints to model protein structures with MODELLER

Protein secondary structure predictions and amino acid long range contact map predictions from primary sequence of proteins have been explored to aid in modelling protein tertiary structures. In order to evaluate the usefulness of secondary structure and 3D-residue contact prediction methods to model protein structures we have used the known Q3 (alpha-helix, beta-strands and irregular turns/loops) secondary structure information, along with residue-residue contact information as restraints for MODELLER. We present here results of our modelling studies on 30 best resolved single domain protein structures of varied lengths. The results shows that it is very difficult to obtain useful models even with 100% accurate secondary structure predictions and accurate residue contact predictions for up to 30% of residues in a sequence. The best models that we obtained for proteins of lengths 37, 70, 118, 136 and 193 amino acid residues are of RMSDs 4.17, 5.27, 9.12, 7.89 and 9.69, respectively. The results show that one can obtain better models for the proteins which have high percent of alpha-helix content. This analysis further shows that MODELLER restrain optimization program can be useful only if we have truly homologous structure(s) as a template where it derives numerous restraints, almost identical to the templates used. This analysis also clearly indicates that even if we satisfy several true residue-residue contact distances, up to 30% of their sequence length with fully known secondary structural information, we end up predicting model structures much distant from their corresponding native structures.     

Use of secondary structural information and C alpha-C alpha distance restraints to model protein structures with MODELLER

Protein secondary structure predictions and amino acid long range contact map predictions from primary sequence of proteins have been explored to aid in modelling protein tertiary structures. In order to evaluate the usefulness of secondary structure and 3D-residue contact prediction methods to model protein structures we have used the known Q3 (alpha-helix,beta-strands and irregular turns/loops) secondary structure information, along with residue-residue contact information as restraints for MODELLER. We present here results of our modelling studies on 30 best resolved single domain protein structures of varied lengths. The results shows that it is very difficult to obtain useful models even with 100% accurate secondary structure predictions and accurate residue contact predictions for up to 30% of residues in a sequence. The best models that we obtained for proteins of lengths 37, 70, 118, 136 and 193 amino acid residues are of RMSDs 4.17, 5.27, 9.12, 7.89 and 9.69,respectively. The results show that one can obtain better models for the proteins which have high percent of alpha-helix content. This analysis further shows that MODELLER restrain optimization program can be useful only if we have truly homologous structure(s) as a template where it derives numerous restraints, almost identical to the templates used. This analysis also clearly indicates that even if we satisfy several true residue-residue contact distances, up to 30%of their sequence length with fully known secondary structural information, we end up predicting model structures much distant from their corresponding native structures.     

GXXXG and AXXXA: common alpha-helical interaction motifs in proteins, particularly in extremophiles

The GXXXG motif is a frequently occurring sequence of residues that is known to favor helix-helix interactions in membrane proteins. Here we show that the GXXXG motif is also prevalent in soluble proteins whose structures have been determined. Some 152 proteins from a non-redundant PDB set contain at least one alpha-helix with the GXXXG motif, 41 +/- 9% more than expected if glycine residues were uniformly distributed in those alpha-helices. More than 50% of the GXXXG-containing alpha-helices participate in helix-helix interactions. In fact, 26 of those helix-helix interactions are structurally similar to the helix-helix interaction of the glycophorin A dimer, where two transmembrane helices associate to form a dimer stabilized by the GXXXG motif. As for the glycophorin A structure, we find backbone-to-backbone atomic contacts of the C alpha-H...O type in each of these 26 helix-helix interactions that display the stereochemical hallmarks of hydrogen bond formation. These glycophorin A-like helix-helix interactions are enriched in the general set of helix-helix interactions containing the GXXXG motif, suggesting that the inferred C alpha-H...O hydrogen bonds stabilize the helix-helix interactions. In addition to the GXXXG motif, some 808 proteins from the non-redundant PDB set contain at least one alpha-helix with the AXXXA motif (30 +/- 3% greater than expected). Both the GXXXG and AXXXA motifs occur frequently in predicted alpha-helices from 24 fully sequenced genomes. Occurrence of the AXXXA motif is enhanced to a greater extent in thermophiles than in mesophiles, suggesting that helical interaction based on the AXXXA motif may be a common mechanism of thermostability in protein structures. We conclude that the GXXXG sequence motif stabilizes helix-helix interactions in proteins, and that the AXXXA sequence motif also stabilizes the folded state of proteins.     

From Principal Component to Direct Coupling Analysis of Coevolution in Proteins: Low-Eigenvalue Modes are Needed for Structure Prediction

Various approaches have explored the covariation of residues in multiple-sequence alignments of homologous proteins to extract functional and structural information. Among those are principal component analysis (PCA), which identifies the most correlated groups of residues, and direct coupling analysis (DCA), a global inference method based on the maximum entropy principle, which aims at predicting residue-residue contacts. In this paper, inspired by the statistical physics of disordered systems, we introduce the Hopfield-Potts model to naturally interpolate between these two approaches. The Hopfield-Potts model allows us to identify relevant ‘patterns’ of residues from the knowledge of the eigenmodes and eigenvalues of the residue-residue correlation matrix. We show how the computation of such statistical patterns makes it possible to accurately predict residue-residue contacts with a much smaller number of parameters than DCA. This dimensional reduction allows us to avoid overfitting and to extract contact information from multiple-sequence alignments of reduced size. In addition, we show that low-eigenvalue correlation modes, discarded by PCA, are important to recover structural information: the corresponding patterns are highly localized, that is, they are concentrated in few sites, which we find to be in close contact in the three-dimensional protein fold.     

Residue-residue contact substitution probabilities derived from aligned three-dimensional structures and the identification of common folds.

We report the derivation of scores that are based on the analysis of residue-residue contact matrices from 443 3-dimensional structures aligned structurally as 96 families, which can be used to evaluate sequence-structure matches. Residue-residue contacts and the more than 3 x 10(6) amino acid substitutions that take place between pairs of these contacts at aligned positions within each family of structures have been tabulated and segregated according to the solvent accessibility of the residues involved. Contact maps within a family of structures are shown to be highly conserved (approximately 75%) even when the sequence identity is approaching 10%. In a comparison involving a globin structure and the search of a sequence databank (> 21,000 sequences), the contact probability scores are shown to provide a very powerful secondary screen for the top scoring sequence-structure matches, where between 69% and 84% of the unrelated matches are eliminated. The search of an aligned set of 2 globins against a sequence databank and the subsequent residue contact-based evaluation of matches locates all 618 globin sequences before the first non-globin match. From a single bacterial serine proteinase structure, the structural template approach coupled with residue-residue contact substitution data lead to the detection of the mammalian serine proteinase family among the top matches in the search of a sequence databank.     

Occurrence of bifurcated three-center hydrogen bonds in proteins

Analysis of 13 high-resolution protein X-ray crystal structures shows that 1204 (24%) of all the 4974 hydrogen bonds are of the bifurcated three-center type with the donor X-H opposing two acceptors A1, A2. They occur systematically in alpha-helices where 90% of the hydrogen bonds are of this type; the major component is (n + 4)N-H ... O = C(n) as expected for a 3.6(13) alpha-helix, and the minor component is (n + 4)N-H ... O = C(n + 1), as observed in 3(10) helices; distortions at the C-termini of alpha-helices are stabilized by three-center bonds. In beta-sheets 40% of the hydrogen bonds are three-centered. The frequent occurrence of three-center hydrogen bonds suggests that they should not be neglected in protein structural studies.     

Deterministic features of side-chain main-chain hydrogen bonds in globular protein structures

A total of 19 835 polar residues from a data set of 250 non-homologous and highly resolved protein crystal structures were used to identify side-chain main-chain (SC-MC) hydrogen bonds. The ratio of the number of SC-MC hydrogen bonds to the total number of polar residues is close to 1:2, indicating the ubiquitous nature of such hydrogen bonds. Close to 56% of the SC-MC hydrogen bonds are local involving side-chain acceptor/donor ('i') and a main-chain donor/acceptor within the window i-5 to i+5. These short-range hydrogen bonds form well defined conformational motifs characterized by specific combinations of backbone and side-chain torsion angles. (a) The Ser/Thr residues show the greatest preference in forming intra-helical hydrogen bonds between the atoms O(gamma)(i) and O(i-4). More than half the examples of such hydrogen bonds are found at the middle of alpha-helices rather than at their ends. The most favoured motif of these examples is alpha(R)alpha(R)alpha(R)alpha(R)(g(-)). (b) These residues also show great preference to form hydrogen bonds between O(gamma)(i) and O(i-3), which are closely related to the previous type and though intra-helical, these hydrogen bonds are more often found at the C-termini of helices than at the middle. The motif represented by alpha(R)alpha(R)alpha(R)alpha(R)(g(+)) is most preferred in these cases. (c) The Ser, Thr and Glu are the most frequently found residues participating in intra-residue hydrogen bonds (between the side-chain and main-chain of the same residue) which are characterized by specific motifs of the form beta(g(+)) for Ser/Thr residues and alpha(R)(g(-)g(+)t) for Glu/Gln. (d) The side-chain acceptor atoms of Asn/Asp and Ser/Thr residues show high preference to form hydrogen bonds with acceptors two residues ahead in the chain, which are characterized by the motifs beta (tt')alphaR and beta(t)alpha(R), respectively. These hydrogen bonded segments, referred to as Asx turns, are known to provide stability to type I and type I' beta-turns. (e) Ser/Thr residues often form a combination of SC-MC hydrogen bonds, with the side-chain donor hydrogen bonded to the carbonyl oxygen of its own peptide backbone and the side-chain acceptor hydrogen bonded to an amide hydrogen three residues ahead in the sequence. Such motifs are quite often seen at the beginning of alpha-helices, which are characterized by the beta(g(+))alpha(R)alpha(R) motif. A remarkable majority of all these hydrogen bonds are buried from the protein surface, away from the surrounding solvent. This strongly indicates the possibility of side-chains playing the role of the backbone, in the protein interiors, to satisfy the potential hydrogen bonding sites and maintaining the network of hydrogen bonds which is crucial to the structure of the protein.