Side-chain structures in the first turn of the alpha-helix

The first three residues at the N terminus of the alpha-helix are called N1, N2 and N3. We surveyed 2102 alpha-helix N termini in 298 high-resolution, non-homologous protein crystal structures for N1, N2 and N3 amino acid and side-chain rotamer propensities and hydrogen-bonding patterns. We find strong structural preferences that are unique to these sites. The rotamer distributions as a function of amino acid identity and position in the helix are often explained in terms of hydrogen-bonding interactions to the free N1, N2 and N3 backbone NH groups. Notably, the "good N2" amino acid residues Gln, Glu, Asp, Asn, Ser, Thr and His preferentially form i, i or i,i+1 hydrogen bonds to the backbone, though this is hindered by good N-caps (Asp, Asn, Ser, Thr and Cys) that compete for these hydrogen bond donors. We find a number of specific side-chain to side-chain interactions between N1 and N2 or between the N-cap and N2 or N3, such as Arg(N-cap) to Asp(N2). The strong energetic and structural preferences found for N1, N2 and N3, which differ greatly from positions within helix interiors, suggest that these sites should be treated explicitly in any consideration of helical structure in peptides or proteins.     

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.     

Molecular dissection,tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans

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.     

Invariant features of globin primary structure and coding of their secondary structure

Invariant features of the primary structure of 67 globins are analysed. These features may be responsible for the formation of the secondary structure of these proteins at the first stage of self-organization (in the unfolded chain). It is shown that in primary structures of globins there are 11 sites or regions of one to four residues in which at least one of the residues Asn, Asp, His, Pro, Ser or Thr is located in every globin (haem-linking His residues are excluded from these sites). An unambiguous correlation exists between the position of these regions and the secondary structure of globins: all these regions (except one) are located near the ends of helices in globins whose three-dimensional structure is known and the ends of all helices (except for the helix F) are coded by such regions. A decrease in the set of residues listed above leads to a sharp drop in the number of regions invariantly occupied by the residues, while an addition of residues such as Tyr and Gly to this set does not eventually increase the number of invariant regions. Five residues (Asn, Asp, His, Ser and Thr) of the six that code the ends of helical regions have polar side groups with a small number of degrees of freedom capable of forming hydrogen bonds with atoms of the backbone with a relatively small loss of entropy. One residue (Pro) has no NH-group and, therefore, has less chance of participating in the formation of hydrogen bonds between atoms of the backbone. This corroborates the hypothesis that competition between hydrogen bonds of short polar side groups and hydrogen bonds in the backbone is essential for the formation of the secondary structure in unfolded protein chains. Amino acid replacements in hydrophobic cores of the 67 globins are considered in the Appendix.     

Effect of the N2 residue on the stability of the α-helix for all 20 amino acids

N2 is the second position in the alpha-helix. All 20 amino acids were placed in the N2 position of a synthetic helical peptide (CH(3)CO-[AXAAAAKAAAAKAAGY]-NH(2)) and the helix content was measured by circular dichroism spectroscopy at 273K. The dependence of peptide helicity on N2 residue identity has been used to determine a free-energy scale by analysis with a modified Lifson-Roig helix-coil theory that includes a parameter for the N2 energy (n2). The rank order of DeltaDeltaG((relative to Ala)) is Glu(-), Asp(-) > Ala > Glu(0), Leu, Val, Gln, Thr, Ile, Ser, Met, Asp(0), His(0), Arg, Cys, Lys, Phe > Asn, > Gly, His(+), Pro, Tyr. The results correlate very well with N2 propensities in proteins, moderately well with N1 and helix interior preferences, and not at all with N-cap preferences. The strongest energetic effects result from interactions with the helix dipole, which favors negative charges at the helix N terminus. Hydrogen bonds to side chains at N2, such as Gln, Ser, and Thr, are weak, despite occurring frequently in protein crystal structures, in contrast to the N-cap position. This is because N-cap hydrogen bonds are close to linear, whereas N2 hydrogen bonds have poor geometry. These results can be used to modify protein stability rationally, help design helices, and improve prediction of helix location and stability.     

Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)     

A propensity scale for type II polyproline helices (PPII): aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline-aromatic interactions

Type II polyproline helices (PPII) are a fundamental secondary structure of proteins, common in globular and nonglobular regions and important in cellular signaling. We developed a propensity scale for PPII using a host-guest system with sequence Ac-GPPXPPGY-NH(2), where X represents any amino acid. We found that proline has the highest PPII propensity, but most other amino acids display significant PPII propensities. The PPII propensity of leucine was the highest of all propensities of non-proline residues. Alanine and residues with linear side chains displayed the next highest PPII propensities. Three classes of residues displayed lower PPII propensities: β-branched amino acids (Thr, Val, and Ile), short amino acids with polar side chains (Asn, protonated Asp, Ser, Thr, and Cys), and aromatic amino acids (Phe, Tyr, and Trp). tert-Leucine particularly disfavored PPII. The basis of the low PPII propensities of aromatic amino acids in this context was significant cis-trans isomerism, with proline-rich peptides containing aromatic residues exhibiting 45-60% cis amide bonds, due to Pro-cis-Pro-aromatic and aromatic-cis-Pro amide bonds.     

On residues in the disallowed region of the Ramachandran map.

An analysis of the occurrence of nonglycyl residues in conformations disallowed in the Ramachandran plot is presented. Ser, Asn, Thr, and Cys have the highest propensities to exhibit such conformations, and the branched aliphatic residues the lowest. Residues cluster in five regions and there are some trends in the types of residues and their side-chain conformations (chi(1)) occupying these. Majority of the residues are found at the edge of helices and strands and in short loops, and are involved in different types of weak, stabilizing interactions. A structural motif has been identified where a residue in disallowed conformation occurs as the first residue of a short 3(10)-helix. On the basis of the types of neighboring residues, the location in the three-dimensional structure and accessibility, there are similarities with the occurrence of cis peptide bonds in protein structures.     

Position dependence of amino acid intrinsic helical propensities II: non-charged polar residues: Ser, Thr, Asn, and Gln.

The assumption that the intrinsic alpha-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by approximately 0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (+/-0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (+/-0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.     

Structures of N-termini of helices in proteins.

We have surveyed 393 N-termini of alpha-helices and 156 N-termini of 3(10)-helices in 85 high resolution, non-homologous protein crystal structures for N-cap side-chain rotamer preferences, hydrogen bonding patterns, and solvent accessibilities. We find very strong rotamer preferences that are unique to N-cap sites. The following rules are generally observed for N-capping in alpha-helices: Thr and Ser N-cap side chains adopt the gauche - rotamer, hydrogen bond to the N3 NH and have psi restricted to 164 +/- 8 degrees. Asp and Asn N-cap side chains either adopt the gauche - rotamer and hydrogen bond to the N3 NH with psi = 172 +/- 10 degrees, or adopt the trans rotamer and hydrogen bond to both the N2 and N3 NH groups with psi = 1-7 +/- 19 degrees. With all other N-caps, the side chain is found in the gauche + rotamer so that the side chain does not interact unfavorably with the N-terminus by blocking solvation and psi is unrestricted. An i, i + 3 hydrogen bond from N3 NH to the N-cap backbone C = O in more likely to form at the N-terminus when an unfavorable N-cap is present. In the 3(10)-helix Asn and Asp remain favorable N-caps as they can hydrogen bond to the N2 NH while in the trans rotamer; in contrast, Ser and Thr are disfavored as their preferred hydrogen bonding partner (N3 NH) is inaccessible. This suggests that Ser is the optimum choice of N-cap when alpha-helix formation is to be encouraged while 3(10)-helix formation discouraged. The strong energetic and structural preferences found for N-caps, which differ greatly from positions within helix interiors, suggest that N-caps should be treated explicitly in any consideration of helical structure in peptides or proteins.     

Secondary structures without backbone: an analysis of backbone mimicry by polar side chains in protein structures.

Backbone mimicry by the formation of closed-loop C7, C10 and C13 (mimics of gamma-, beta- and alpha-turns) conformations through side chain-main chain hydrogen bonds by polar groups is a frequent observation in protein structures. A data set of 250 non-homologous and high-resolution protein crystal structures was used to analyze these conformations for their characteristic features. Seven out of the nine polar residues (Ser, Thr, Asn, Asp, Gln, Glu and His) have hydrogen bonding groups in their side chains which can participate in such mimicry and as many as 15% of all these polar residues engage in such conformations. The distributions of dihedral angles of these mimics indicate that only certain combinations of the dihedral angles involved aid the formation of these mimics. The observed examples were categorized into various classes based on these combinations, resulting in well defined motifs. Asn and Asp residues show a very high capability to perform such backbone secondary structural mimicry. The most highly mimicked backbone structure is of the C10 conformation by the Asx residues. The mimics formed by His, Ser, Thr and Glx residues are also discussed. The role of such conformations in initiating the formation of regular secondary structures during the course of protein folding seems significant.     

The asparagine-stabilized beta-turn of apamin: contribution to structural stability from dynamics simulation and amide hydrogen exchange analysis

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to explore the contribution of hydrogen bonds involving Asn2 to local (beta-turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retained a stable conformation during 2.4 ns of simulation at 67 degrees C, with high beta-turn stability characterized by backbone-side chain hydrogen bonds involving beta-turn residues K4 and A5, with the N2 side chain amide carbonyl. The loss of stabilizing interactions involving the N2 side chain resulted in the loss of the beta-turn conformation in the apamin N2A simulations (27 or 67 degrees C). This loss of beta-turn stability propagates throughout the peptide structure, with destabilization of the C-terminal helix connected to the N-terminal region by two disulfide bonds. Backbone stability in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, loss of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta-turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in apamin N2A as compared with apamin, corresponding to free energy (deltaDeltaG(uf)) of around 1.5 kcal M(-1) at 20 degrees C. This is equivalent to the contribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta-turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in apamin that account for virtually the full thermodynamic stability of the peptide have been quantitated.     

Sequence context strongly modulates association of polar residues in transmembrane helices

Polar residues are capable of mediating the association of membrane-embedded helices through the formation of side-chain/side-chain inter-helical hydrogen bonds. However, the extent to which native van der Waals packing of the residues surrounding the polar locus can enhance, or interfere with, the interaction of polar residues has not yet been studied. We examined the propensities of four polar residues (aspartic acid, asparagine, glutamic acid, and glutamine) to promote self-association of transmembrane (TM) domains in several biologically derived sequence environments, including (i). four naturally occurring TM domains that contain a Glu or Gln residue (Tnf5/CD40 ligand, C79a/Ig-alpha, C79b/Ig-beta, and Fut3/alpha-fucosyltransferase); and (ii). variants of bacteriophage M13 major coat protein TM segment with Asp and Asn at interfacial and non-interfacial positions. Self-association was quantified by the TOXCAT assay, which measures TM helix self-oligomerization in the Escherichia coli inner membrane. While an appropriately placed polar residue was found in several cases to significantly stabilize TM helix-helix interactions through the formation of an interhelical hydrogen bond, in other cases the strongly polar residues did not enhance the association of the two helices. Overall, these results suggest that an innate structural mechanism may operate to control non-specific association of membrane-embedded polar residues.     

Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis.

The proposed roles of Cys148 and Asp179 in deoxycytidylate (dCMP) hydroxymethylase (CH) have been tested using site-directed mutagenesis. CH catalyzes the formation of 5-(hydroxymethyl)-dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate. CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. Conversion of Cys148 to Asp, Gly, or Ser decreases CH activity at least 10(5)-fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys residue in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of the substrate dUMP to the side-chain amide of an Asn; the corresponding residue in CH is Asp179. Conversion of Asp179 to Asn reduces the value of kcat/KM for dCMP by (1.5 x 10(4))-fold and increases the value of kcat/KM for dUMP by 60-fold; as a result, CH(D179N) has a slight preference for dUMP. Wild-type CH and CH(D179N) are covalently inactivated by 5-fluoro-dUMP, a mechanism-based inactivator of TS. Asp179 is proposed to stabilize covalent catalytic intermediates, by protonating N3 of the pyrimidine-CH adduct.     

Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs.     

A Ser residue influences the structure and stability of a Pro-kinked transmembrane helix dimer

When localized adjacent to a Pro-kink, Thr and Ser residues can form hydrogen bonds between their polar hydroxyl group and a backbone carbonyl oxygen and thereby modulate the actual bending angle of a distorted transmembrane α-helix. We have used the homo-dimeric transmembrane cytochrome b(559)' to analyze the potential role of a highly conserved Ser residue for assembly and stabilization of transmembrane proteins. Mutation of the conserved Ser residue to Ala resulted in altered heme binding properties and in increased stability of the holo-protein, most likely by tolerating subtle structural rearrangements upon heme binding. The results suggest a crucial impact of an intrahelical Ser hydrogen bond in defining the structure of a Pro-kinked transmembrane helix dimer.     

Side-chain flexibility in proteins upon ligand binding

Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side-chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino-acid side-chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side-chain rearrangements upon ligand binding. We constructed two non-redundant databases (980 and 353 entries) of "paired" protein structures in complexed (holo-protein) and uncomplexed (apo-protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side-chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., approximately 85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys > Arg, Gln, Met > Glu, Ile, Leu > Asn, Thr, Val, Tyr, Ser, His, Asp > Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side-chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets.     

Conformation-Determining role for the N-acetyl group in the O-glycosidic linkage, α-GalNAc-Thr

An ¹H-nmr study of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactopyranose (AcGalNAc) glycosylated Thr-containing tripeptides in Me₂SO-d₆ solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z-Thr(AcGalNAc)-Ala-Ala-OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N-acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac-Thr(AcGalNAc)-Ala-Ala-OMe, a different intramolecular hydrogen bond between the sugar N-acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O-glycoproteins.     

Dissecting α-helices: Position-specific analysis of α-helices in globular proteins

An analysis of the amino acid distributions at 15 positions, viz., N“, N′, Ncap, N1, N2, N3, N4, Mid, C4, C3, C2, C1, Ccap, C′, and C” in 1,131 α-helices reveals that each position has its own unique characteristics. In general, natural helix sequences optimize by identifying the residues to be avoided at a given position and minimizing the occurrence of these avoided residues rather than by maximizing the preferred residues at various positions. Ncap is most selective in its choice of residues, with six amino acids (S, D, T, N, G, and P) being preferred at this position and another 11 (V, I, F, A, K, L, Y, R, E, M, and Q) being strongly avoided. Ser, Asp, and Thr are all more preferred at Ncap position than Asn, whose role at helix N-terminus has been highlighted by earlier analyses. Furthermore, Asn is also found to be almost equally preferred at helix C-terminus and a novel structural motif is identified, involving a hydrogen bond formed by N^δ2 of Asn at Ccap or C1 position, with the backbone carbonyl oxygen four residues inside the helix. His also forms a similar motif at the C-terminus. Pro is the most avoided residue in the main body (N4 to C4 positions) and at C-ter-minus, including Ccap of an α-helix. In 1,131 α-helices, no helix contains Pro at C3 or C2 positions. However, Pro is highly favoured at N1 and C′. The doublet X-Pro, with Pro at C′ position and extended backbone conformation for the X residue at Ccap, appears to be a common structural motif for termination of α-helices, in addition to the Schellman motif. Main body of the helix shows a high preference for aliphatic residues Ala, Leu, Val, and Ile, while these are avoided at helix termini. A propensity scale for amino acids to occur in the middle of helices has been obtained. Comparison of this scale with several previously reported scales shows that this scale correlates best with the experimentally determined values. Proteins 31:460–476, 1998. © 1998 Wiley-Liss, Inc.     

Long-range nature of the interactions between titratable groups in Bacillus agaradhaerens family 11 xylanase: pH titration of B. agaradhaerens xylanase

Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan. The protonation behavior of titratable groups of the uniformly (15)N- and (13)C-labeled xylanase was investigated by multinuclear NMR spectroscopy. A total of 224 chemical shift titration curves corresponding to (1)H, (13)C, and (15)N resonances revealed pK(a) values for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues. Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment. Subsite -1 containing the catalytic dyad shows a long-range interaction over 9 A with Asp21 via two hydrogen bonds with Asn45 as the mediator. This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate. The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking. Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pK(a) values of the nearby acidic side chains. However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH approximately 6 and/or an overall change in the hydrogen network in the substrate binding cleft.