Hydrogen bonds between short polar side chains and peptide backbone: prevalence in proteins and effects on helix-forming propensities

A survey of 322 proteins showed that the short polar (SP) side chains of four residues, Thr, Ser, Asp, and Asn, have a very strong tendency to form hydrogen bonds with neighboring backbone amides. Specifically, 32% of Thr, 29% of Ser, 26% of Asp, and 19% of Asn engage in such hydrogen bonds. When an SP residue caps the N terminal of a helix, the contribution to helix stability by a hydrogen bond with the amide of the N3 or N2 residue is well established. When an SP residue is in the middle of a helix, the side chain is unlikely to form hydrogen bonds with neighboring backbone amides for steric and geometric reasons. In essence the SP side chain competes with the backbone carbonyl for the same hydrogen-bonding partner (i.e., the backbone amide) and thus SP residues tend to break backbone carbonyl-amide hydrogen bonds. The proposition that this is the origin for the low propensities of SP residues in the middle of alpha helices (relative to those of nonpolar residues) was tested. The combined effects of restricting side-chain rotamer conformations (documented by Creamer and Rose, Proc Acad Sci USA, 1992;89:5937-5941; Proteins, 1994;19:85-97) and excluding side- chain to backbone hydrogen bonds by the helix were quantitatively analyzed. These were found to correlate strongly with four experimentally determined scales of helix-forming propensities. The correlation coefficients ranged from 0.72 to 0.87, which are comparable to those found for nonpolar residues (for which only the loss of side-chain conformational entropy needs to be considered).     

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.     

Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis.     

Molecular dynamics simulations of the unfolding of an alpha-helical analogue of ribonuclease A S-peptide in water

Molecular dynamics simulations of the S-peptide analogue AETAAAKFLREHMDS have been conducted in aqueous solution for 300 ps at 278 K and for 500 ps in two different runs at 358 K. The results show agreement with experimental observations in that at low temperature, 5 degrees C, the helix is stable, while unfolding is observed at 85 degrees C. In the low-temperature simulation a solvent-separated ion pair was formed between Glu-2 and Arg-10, and the side chain of His-12 reoriented toward the C-terminal end of the alpha-helix. Detailed analyses of the unfolding pathways at high temperature have also revealed that the formation or disappearance of main-chain helical hydrogen bonds occurs frequently through an alpha in equilibrium with 3(10) in equilibrium with no hydrogen bond sequence.     

Variability in the backbone conformation of cyclic pentapeptides. Crystal structure of cyclic(Gly-L-Pro-D-Phe-Gly-L-Ala)

All the peptide bonds in cyclic(Gly-L-Pro-D-Phe-Gly-L-Ala) are in the trans conformation; however, the peptide bond C'5-N1 is twisted by 19 degrees from planarity (omega 5 = -161 degrees). A Type II beta-turn encompasses the L-Pro-D-Phe residues. Carbonyl oxygens O2, O4 and O5 are directed to the same side of the average plane through the backbone ring and they form hydrogen bonds with N3, N5 and N1, respectively, in adjacent molecules in a stacked column where the adjacent molecules are related by one translational unit. The conformation of the backbone is different from that established in other molecules with the DLDDL chirality sequence. The P21 cell contains two molecules of C21H26N5O5 with a = 4.836(2) A, b = 18.346(8) A, c = 12.464(5) A and beta = 100.05(4) degrees. The R factor for 1382 data with [F0[ greater than 1 sigma is 7.0%.     

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.     

Hydrogen bonding in helical polypeptides from molecular dynamics simulations and amide hydrogen exchange analysis: alamethicin and melittin in methanol.

Molecular dynamics simulations of ion channel peptides alamethicin and melittin, solvated in methanol at 27 degrees C, were run with either regular alpha-helical starting structures (alamethicin, 1 ns; melittin 500 ps either with or without chloride counterions), or with the x-ray crystal coordinates of alamethicin as a starting structure (1 ns). The hydrogen bond patterns and stabilities were characterized by analysis of the dynamics trajectories with specified hydrogen bond angle and distance criteria, and were compared with hydrogen bond patterns and stabilities previously determined from high-resolution NMR structural analysis and amide hydrogen exchange measurements in methanol. The two alamethicin simulations rapidly converged to a persistent hydrogen bond pattern with a high level of 3(10) hydrogen bonding involving the amide NH's of residues 3, 4, 9, 15, and 18. The 3(10) hydrogen bonds stabilizing amide NH's of residues C-terminal to P2 and P14 were previously proposed to explain their high amide exchange stabilities. The absence, or low levels of 3(10) hydrogen bonds at the N-terminus or for A15 NH, respectively, in the melittin simulations, is also consistent with interpretations from amide exchange analysis. Perturbation of helical hydrogen bonding in the residues before P14 (Aib10-P14, alamethicin; T11-P14, melittin) was characterized in both peptides by variable hydrogen bond patterns that included pi and gamma hydrogen bonds. The general agreement in hydrogen bond patterns determined in the simulations and from spectroscopic analysis indicates that with suitable conditions (including solvent composition and counterions where required), local hydrogen-bonded secondary structure in helical peptides may be predicted from dynamics simulations from alpha-helical starting structures. Each peptide, particularly alamethicin, underwent some large amplitude structural fluctuations in which several hydrogen bonds were cooperatively broken. The recovery of the persistent hydrogen bonding patterns after these fluctuations demonstrates the stability of intramolecular hydrogen-bonded secondary structure in methanol (consistent with spectroscopic observations), and is promising for simulations on extended timescales to characterize the nature of the backbone fluctuations that underlie amide exchange from isolated helical polypeptides.     

Helix bending in alamethicin: molecular dynamics simulations and amide hydrogen exchange in methanol

Molecular dynamics simulations of alamethicin in methanol were carried out with either a regular alpha-helical conformation or the x-ray crystal structure as starting structures. The structures rapidly converged to a well-defined hydrogen-bonding pattern with mixed alpha-helical and 3(10)-helical hydrogen bonds, consistent with NMR structural characterization, and did not unfold throughout the 1-ns simulation, despite some sizable backbone fluctuations involving reversible breaking of helical hydrogen bonds. Bending of the helical structure around residues Aib10-Aib13 was associated with reversible flips of the peptide bonds involving G11 (Aib10-G11 or G11-L12 peptide bonds), yielding discrete structural states in which the Aib10 carbonyl or (rarely) the G11 carbonyl was oriented away from the peptide helix. These peptide bond reversals could be accommodated without greatly perturbing the adjacent helical structure, and intramolecular hydrogen bonding was generally maintained in bent states through the formation of new (non-alpha or 3[10]) hydrogen bonds with good geometries: G11 NH-V9 CO (inverse gamma turn), Aib13 NH-Aib8 CO (pi-helix) and, rarely, L12 NH- Q7 NH (pi-helix). These observations may reconcile potentially conflicting NMR structural information for alamethicin in methanol, in which evidence for conformational flexibility in the peptide sequence before P14 (G11-Aib13) contrasts with the stability of backbone amide NH groups to exchange with solvent. Similar reversible reorientation of the Thr11-Gly12 peptide bond of melittin is also observed in dynamics simulations in methanol (R. B. Sessions, N. Gibbs, and C. E. Dempsey, submitted). This phenomenon may have some role in the orientation of the peptide carbonyl in solvating the channel lumen in membrane ion channel states of these peptides.     

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove. The side chain at P9 is clearly the one that contributes the most to the thermal stability of the MHC-peptide complexes, as destabilization up to 25 degrees C are obtained after P9 mutation. Interestingly, structure-stability relationships do not fully mirror structure-binding relationships. As important as the C-terminal side chain are the terminal ammonium and carboxylate groups. Removal of a single H-bond between HLA-B27 and the terminal peptide moieties results in thermal destabilization up to 10 degrees C. Depending on the bound peptide and the location of the deleted H-bond, the decrease in the thermal stability of the corresponding complex is quantitatively different. The present study suggests that any peptidic amino acid at positions 3 and 9 promotes refolding of the B27-peptide complex. Once the complex is formed, the C-terminal side chain seems to play an important role for maintaining a stable complex.     

Structure and dynamics of an amphiphilic peptide in a lipid bilayer: a molecular dynamics study

A molecular dynamics simulation of a simple model membrane system composed of a single amphiphilic helical peptide (ace-K2GL16K2A-amide) in a fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer was performed for a total of 1060 ps. The secondary structure of the peptide and its stability were described in terms of average dihedral angles, phi and psi, and the C alpha torsion angles formed by backbone atoms; by the average translation per residue along the helix axis; and by the intramolecular peptide hydrogen bonds. The results indicated that residues 6 through 15 remain in a stable right-handed alpha-helical conformation, whereas both termini exhibit substantial fluctuations. A change in the backbone dihedral angles for residues 16 and 17 is accompanied by the loss of two intramolecular hydrogen bonds, leading to a local but long-lived disruption of the helix. The dynamics of the peptide was characterized in terms of local and global helix motions. The local motions of the N-H bond angles were described in terms of the autocorrelation functions of P2[cos thetaNH(t, t + tau)] and reflected the different degrees of local peptide order as well as a variation in time scale for local motions. The chi1 and chi2 dihedral angles of the leucine side chains underwent frequent transitions between potential minima. No connection between the side-chain positions and their mobility was observed, however. In contrast, the lysine side chains displayed little mobility during the simulation. The global peptide motions were characterized by the tilting and bending motions of the helix. Although the peptide was initially aligned parallel to the bilayer normal, during the simulation it was observed to tilt away from the normal, reaching an angle of approximately 25 degrees by the end of the simulation. In addition, a slight bend of the helix was detected. Finally, the solvation of the peptide backbone and side-chain atoms was also investigated.     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.     

Synthesis and crystal structures of Boc-L-Asn-L-Pro-OBzl.CH3OH and dehydration side product, Boc-beta-cyano-L-alanine-L-Pro-OBzl

Boc-L-Asn-L-Pro-OBzl: C21H29O6N3.CH3OH, Mr = 419.48 + CH3 OH, monoclinic, P2(1), a = 10.049(1), b = 10.399(2), c = 11.702(1) A, beta = 92.50(1)degrees, V = 1221.7(3) A3, dx = 1.14 g.cm-3, Z = 2, CuK alpha (lambda = 1.54178 A), F(000) = 484 (with solvent), 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1745, R = 0.043, Rw = 0.062, S = 1.66. Boc-beta-cyano-L-alanine-L-Pro-OBzl: C21H27O5N3, Mr = 401.46, orthorhombic, P2(1)2(1)2(1), a = 15.741(3), b = 21.060(3), c = 6.496(3) A, V = 2153(1) A3, dx = 1.24 g.cm-3, Z = 4, CuK alpha (lambda = 1.54178 A), F(000) = 856, 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1573, R = 0.055, Rw = 0.078, S = 1.86. The tert.-butyloxycarbonyl (Boc) protected dipeptide benzyl ester (OBzl), Boc-L-Asn-L-Pro-OBzl, prepared from a mixed anhydride reaction using isobutylchloroformate, Boc-L-asparagine, and HCl.L-proline-OBzl, crystallized with one methanol per asymmetric unit in an extended conformation with the Asn-Pro peptide bond trans. Intermolecular hydrogen bonding occurs between the methanol and the Asn side chain and between the peptide backbone and the Asn side chain. A minor impurity due to the dehydration of the Asn side chain to a beta-CNala crystallized with a similar extended conformation and a single intermolecular hydrogen bond.     

Salt, phosphate and the Bohr effect at the hemoglobin beta chain C terminus studied by hydrogen exchange

Hydrogen exchange experiments using functional labeling and fragment separation methods were performed to study interactions at the C terminus of the hemoglobin beta subunit that contribute to the phosphate effect and the Bohr effect. The results show that the H-exchange behavior of several peptide NH at the beta chain C terminus is determined by a transient, concerted unfolding reaction involving five or more residues, from the C-terminal His146 beta through at least Ala142 beta, and that H-exchange rate can be used to measure the stabilization free energy of interactions, both individually and collectively, at this locus. In deoxy hemoglobin at pH 7.4 and 0 degrees C, the removal of 2,3-diphosphoglycerate (DPG) or pyrophosphate (loss of a salt to His143 beta) speeds the exchange of the beta chain C-terminal peptide NH protons by 2.5-fold (at high salt), indicating a destabilization of the C-terminal segment by 0.5 kcal of free energy. Loss of the His146 beta 1 to Asp94 beta 1 salt link speeds all these protons by 6.3-fold, indicating a bond stabilization free energy of 1.0 kcal. When both these salt links are removed together, the effect is found to be strictly additive; all the protons exchange faster by 16-fold indicating a loss of 1.5 kcal in stabilization free energy. Added salt is slightly destabilizing when DPG is present but provides some increased stability, in the 0.2 kcal range, when DPG is absent. The total allosteric stabilization energy at each beta chain C terminus in deoxy hemoglobin under these conditions is measured to be 3.8 kcal (pH 7.4, 0 degrees C, with DPG). In oxy hemoglobin at pH 7.4 and 0 degrees C, stability at the beta chain C terminus is essentially independent of salt concentration, and the NES modification, which in deoxy hemoglobin blocks the His146 beta to Asp94 beta salt link, has no destabilizing effect, either at high or low salt. These results appear to show that the His146 beta salt link, which participates importantly in the alkaline Bohr effect, does not reform to Asp94 beta or to any other salt link acceptor in a stable way in oxy hemoglobin at low or high salt conditions.     

pH dependence of hydrogen exchange from backbone peptide amides in apamin

The kinetics of hydrogen exchange of the 11 most protected backbone amides of bee venom apamin have been measured between pH 1 and pH 8.5 by using time-resolved and saturation-transfer NMR spectroscopy. The five amides most protected from base-catalyzed exchange, those of residues 5 and 12-15, show highly correlated exchange behavior in the base-catalyzed regime. It is proposed that the intramolecular hydrogen bonds stabilizing these amides define a stable cooperative unit of secondary structure in apamin (a C-terminal helix and an N-terminal beta-turn). This conformational unit is further stabilized (by 5-6 kJ mol-1) on titration of the Glu-7 side-chain carboxyl group. The relative contributions of specific intramolecular interactions to this conformational stabilization are estimated. The pHminima in the pH-dependent single amide exchange curves are compared with values predicted by correcting for sequence-dependent contributions to amide exchange rates [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. The lack of correlation suggests that the "open" conformers from which amide exchange occurs are nonrandom. This conclusion is dependent on the assumption that acid-catalyzed exchange occurs via N-protonation so that residual conformational effects on exchange rates in the open conformers will affect acid- and base-catalyzed rates in approximately equal and opposite ways. A strong correlation between the measured pHminima and the amide proton chemical shifts is observed, however, and this may be most easily accommodated if acid-catalyzed exchange occurs by the imidic acid mechanism (via amide O-protonation).     

Modular design of synthetic protein mimics. Characterization of the helical conformation of a 13-residue peptide in crystals

The incorporation of alpha-aminoisobutyryl (Aib) residues into peptide sequences facilitates helical folding. Aib-containing sequences have been chosen for the design of rigid helical segments in a modular approach to the construction of a synthetic protein mimic. The helical conformation of the synthetic peptide Boc-Aib-(Val-Ala-Leu-Aib)3-OMe in crystals is established by X-ray diffraction. The 13-residue apolar peptide adopts a helical form in the crystal with seven alpha-type hydrogen bonds in the middle and 3(10)-type hydrogen bonds at either end. The helices stack in columns, zigzag rather than linear, by means of direct NH...OC head to tail hydrogen bonds. Leucyl side chains are extended on one side of the helix and valyl side chains on the other side. Water molecules form hydrogen bonds with several backbone carbonyl oxygens that also participate in alpha-helix hydrogen bonds. There is no apparent distortion of the helix caused by hydration. The space group is P2(1)2(1)2(1), with a = 9.964 (3) A, b = 20.117 (3) A, c = 39.311 (6) A, Z = 4, and dx = 1.127 g/cm3 for C64H106N13O16.1.33H2O. The final agreement factor R was 0.089 for 3667 data observed greater than 3 sigma(F) with a resolution of 0.9 A.     

Asparagine-mediated self-association of a model transmembrane helix

In membrane proteins, the extent to which polarity, hydrogen bonding, and van der Waals packing interactions of the buried, internal residues direct protein folding and association of transmembrane segments is poorly understood. The energetics associated with these various interactions should differ substantially between membrane versus water-soluble proteins. To help evaluate these energetics, we have altered a water-soluble, two-stranded coiled-coil peptide to render its sequence soluble in membranes. The membrane-soluble peptide associates in a monomer-dimer-trimer equilibrium, in which the trimer predominates at the highest peptide/detergent ratios. The oligomers are stabilized by a buried Asn side chain. Mutation of this Asn to Val essentially eliminates oligomerization of the membrane-soluble peptide. Thus, within a membrane-like environment, interactions involving a polar Asn side chain provide a strong thermodynamic driving force for membrane helix association.     

Spatial structure of apamin in solution]

The calculation of the complete spatial structure of the bee venom peptide neurotoxin apamin has been carried out by means of a method elaborated earlier. It is based on the joint utilization of the molecular mechanics algorithms and NMR spectroscopy data. It was established that the molecule backbone conformation in solution may be represented as the combination of the beta-turn III (residues 2-5) and alpha-helical segment (9-18) both separated by the non-standard bend IV (5-8). The most probable system of the intramolecular hydrogen bonds in the apamin polypeptide backbone was proposed. Certain amino acid residues have been shown to be characterized by the lack of strict determination of the conformations of their side chains which may be realized in a few states providing approximately equal stabilization of the same form of the main chain. The conformational parameters of the proposed apamin structural model are appropriate to the NMR spectroscopy data derived from the literature and used in the calculations and are not contradictory to other experimental information.     

Helix-forming tendencies of amino acids depend on the restrictions of side-chain rotamer conformations: crystal structure of the tripeptide GAI in two crystalline forms.

In our attempts to design crystalline alpha-helical peptides, we synthesized and crystallized GAI (C11H21N3O4) in two crystal forms, GAI1 and GAI2. Form 1 (GAI1) Gly-L-Ala-L-Ile (C11H21N3O4.3H2O) crystals are monoclinic, space group P2(1) with a = 8.171(2), b = 6.072(4), c = 16.443(4) A, beta = 101.24(2) degrees, V = 800 A3, Dc = 1.300 g cm-3 and Z = 2, R = 0.081 for 482 reflections. Form 2 (GAI2) Gly-L-Ala-L-Ile (C11H21N3O4.1/2H2O) is triclinic, space group P1 with a = 5.830(1), b = 8.832(2), c = 15.008(2) A, alpha = 102.88(1), beta = 101.16(2), gamma = 70.72(2) degrees, V = 705 A3, Z = 2, Dc = 1.264 g cm-3, R = 0.04 for 2582 reflections. GAI1 is isomorphous with GAV and forms a helix, whereas GAI2 does not. In GAI1, the tripeptide molecule is held in a near helical conformation by a water molecule that bridges the NH3+ and COO- groups, and acts as the fourth residue needed to complete the turn by forming two hydrogen bonds. Two other water molecules form intermolecular hydrogen bonds in stabilizing the helical structure so that the end result is a column of molecules that looks like an incipient alpha-helix. GAI2 imitates a cyclic peptide and traps a water molecule. The conformation angles chi 11 and chi 12 for the side chain are (-63.7 degrees, 171.1 degrees) for the helical GAI1, and (-65.1 degrees, 58.6 degrees) and (-65.0 degrees, 58.9 degrees) for the two independent nonhelical molecules in GAI2; in GAI1, both the C gamma atoms point away from the helix, whereas in GAI2 the C gamma atom with the g+ conformation points inward to the helix and causes sterical interaction with atoms in the adjacent peptide plane. From these results, it is clear that the helix-forming tendencies of amino acids correlate with the restrictions of side-chain rotamer conformations. Both the peptide units in GAI1 are trans and show significant deviation from planarity [omega 1 = -168(1) degrees; omega 2 = -171(1) degrees] whereas both the peptide units in both the molecules A and B in GAI2 do not show significant deviation from planarity [omega 1 = 179.3(3) degrees; omega 2 = -179.3(3) degrees for molecule A and omega 1 = 179.5(3) degrees; omega 2 = -179.4(3) degrees for molecule B], indicating that the peptide planes in these incipient alpha-helical peptides are considerably bent.     

Sequence and structure patterns in proteins from an analysis of the shortest helices: implications for helix nucleation

The shortest helices (three-length 3(10) and four-length alpha), most abundant among helices of different lengths, have been analyzed from a database of protein structures. A characteristic feature of three-length 3(10)-helices is the shifted backbone conformation for the C-terminal residue (phi,psi angles: -95 degrees,0 degrees ), compared to the rest of the helix (-62 degrees,-24 degrees ). The deviation can be attributed to the release of electrostatic repulsion between the carbonyl oxygen atoms at the two C-terminal residues and further stabilization (due to a more linear geometry) of an intrahelical hydrogen bond. A consequence of this non-canonical C-terminal backbone conformation can be a potential origin of helix kinks when a 3(10)-helix is sequence-contiguous at the alpha-helix N-terminal. An analysis of hydrogen bonding, as well as hydrophobic interactions in the shortest helices shows that capping interactions, some of them not observed for longer helices, dominate at the N termini. Further, consideration of the distribution of amino acid residues indicates that the shortest helices resemble the N-terminal end of alpha-helices rather than the C terminus, implying that the folding of helices may be initiated at the N-terminal end, which does not get propagated in the case of the shortest helices. Finally, pairwise comparison of beta-turns and the shortest helices, based on correlation matrices of site-specific amino acid composition, and the relative abundance of these short secondary structural elements, leads to a helix nucleation scheme that considers the formation of an isolated beta-turn (and not an alpha-turn) as the helix nucleation step, with shortest 3(10)-helices as intermediates between the shortest alpha-helix and the beta-turn. Our results ascribe an important role played by shortest 3(10)-helices in proteins with important structural and folding implications.     

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.