Helix folding simulations with various initial conformations.

Using a solvent-referenced energy calculation, a 16-residue peptide with alanine side chains folded into predominantly alpha-helical conformations during constant temperature (274 K) simulations. From different initial conformations, helical conformations were reached and the multiple energy minima did not become a serious problem. Under the same conditions, the simulation did not indiscriminately fold a sequence such as polyglycine into stable helices. Interesting observations from the simulations relate to the folding mechanism. The electrostatic interactions between the successive amides favored extended conformations (or beta strands) and caused energy barriers to helix folding. beta-bends were observed as intermediates during helix nucleation. The helix propagation toward the C-terminus seemed faster than that toward the N-terminus. In helical conformations, hydrogen bond oscillation between the i,i+ 4 and the i,i+3 patterns was observed. The i,i+3 hydrogen bonds occurred more frequently during helix propagation and deformation near both ends of the helical segment.     

The sequence TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase displays structural ambivalence and interconverts between alpha-helical and beta-hairpin conformations mediated by collapsed conformational states.

The peptide TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase adopts a helical conformation in the crystal structure and is a site for two hydrated helical segments, which are thought to be helical folding intermediates. Overlapping sequences of four to five residues from the peptide, sample both helical and strand conformations in known protein structures, which are dissimilar to glyceraldehyde-3-phosphate dehydrogenase suggesting that the peptide may have a structural ambivalence. Molecular dynamics simulations of the peptide sequence performed for a total simulation time of 1.2 micros, starting from the various initial conformations using GROMOS96 force field under NVT conditions, show that the peptide samples a large number of conformational forms with transitions from alpha-helix to beta-hairpin and vice versa. The peptide, therefore, displays a structural ambivalence. The mechanism from alpha-helix to beta-hairpin transition and vice versa reveals that the compact bends and turns conformational forms mediate such conformational transitions. These compact structures including helices and hairpins have similar hydrophobic radius of gyration (Rgh) values suggesting that similar hydrophobic interactions govern these conformational forms. The distribution of conformational energies is Gaussian with helix sampling lowest energy followed by the hairpins and coil. The lowest potential energy of the full helix may enable the peptide to take up helical conformation in the crystal structure of the glyceraldehyde-3-phosphate dehydrogenase, even though the peptide has a preference for hairpin too. The relevance of folding and unfolding events observed in our simulations to hydrophobic collapse model of protein folding are discussed.     

Thermodynamics and mechanism of alpha helix initiation in alanine and valine peptides

We used molecular dynamics simulations to study the folding/unfolding of one of turn of an alpha helix in Ac-(Ala)3-NHMe and Ac-(Val)3-NHMe. Using specialized sampling techniques, we computed free energy surfaces as functions of a conformational coordinate that corresponds to alpha helices at small values and to extended conformations at large values. Analysis of the peptide conformations populated during the simulations showed that alpha helices, reverse turns, and extended conformations correspond to minima on the free energy surfaces of both peptides. The free energy difference between alpha helix and extended conformations, determined from the equilibrium constants for helix unfolding, is approximately -1 kcal/mol for Ac-(Ala)3-NHMe and -5 kcal/mol for Ac-(Val)3-NHMe. The mechanism observed in our simulations, which includes reverse turns as important intermediates along the helix folding/unfolding pathway, is consistent with a mechanism proposed previously. Our results predict that both peptides (but especially the Ala peptide) have a much larger equilibrium constant for helix initiation than is predicted by the helix-coil transition theory with the host-guest parameters. We also predict a much greater difference in the equilibrium constants than the theory predicts. Insofar as helix initiation is concerned, our results suggest that the large difference between the helical propensities of Ala and Val cannot be explained by simple concepts such as side-chain rotamer restriction or unfavorable steric interactions. Rather, the origin of the difference appears to be quite complicated because it involves subtle differences in the solvation of the two peptides. The two peptides have similar turn-extended equilibria but very different helix-turn equilibria, and the difference in helical propensities reflects the fact that the helix-turn equilibrium strongly favors the turns in Ac-(Val)3-NHMe, while it favors the helices in Ac-(Ala)3-NHMe. We also computed thermodynamic decompositions of the free energy surfaces, and these revealed that the helix-turn equilibria are vastly different primarily because the changes in peptide-water interactions that accompany helix-to-turn conformational changes are qualitatively different for the two peptides.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Unfolding of an alpha-helix in water.

We describe a 1 ns molecular dynamics simulation of an 18-residue peptide (corresponding to a portion of the H helix of myoglobin) in water. The initial helical conformation progressively frays to a more disordered structure, with the loss of internal secondary structure generally proceeding from the C-terminus toward the N-terminus. Although a variety of mechanisms are involved in the breaking of helical hydrogen bonds, the formation of transient turn structures, with i----i + 3 hydrogen bonds, and bifurcated hydrogen-bond structures intermediate between alpha and turn or 3(10) structures is a common motif. In some cases a single water molecule is inserted into an internal hydrogen bond, but it is also common to have several water molecules involved in transient intermediates. Overall, the results provide new information about the detailed mechanisms by which helices are made and broken in aqueous solution.     

1H NMR studies of the solution conformations of an analogue of the C-peptide of ribonuclease A

Two-dimensional NMR experiments have been performed on a peptide, succinyl-AE-TAAAKFLRAHA-NH2, related to the amino-terminal sequence of ribonuclease A. This peptide contains 50-60% helix in 0.1 M NaCl solution, pH 5.2, 3 degrees C, as measured by circular dichroism. NOESY spectra of the peptide in aqueous solution at low temperatures show a number of NOE connectivities that are used to determine the highly populated conformations of the peptide in solution. Short-range dNN(i, i + 1) and d alpha N(i, i + 1) connectivities and medium-range d alpha beta(i, i + 3) and d alpha N(i, i + 3) connectivities are detected. The pattern of NOE connectivities unambiguously establishes the presence of helix in this peptide. The magnitudes of the 3JHN alpha coupling constants and the intensities of the dNN(i, i + 1) and d alpha N(i,i + 1) NOEs allow the evaluation of the position of the helix along the peptide backbone. These data indicate that the amino terminus of the peptide is less helical than the remainder of the peptide. The observation of several long-range NOEs that are atypical of helices indicates the presence of a high population of peptide molecules in which the first three residues are distorted out of the helical conformation. The absence of these NOEs in a related peptide, RN-31, in which Arg 10 has been changed to Ala, suggests that this distortion at the amino-terminal end of the peptide arises from the formation of a salt bridge between Glu 2 and Arg 10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Helices in peptoids of alpha- and beta-peptides

Peptoids of alpha- and beta-peptides (alpha- and beta-peptoids) can be obtained by shifting the amino acid side chains from the backbone carbon atoms of the monomer constituents to the peptide nitrogen atoms. They are, therefore, N-substituted poly-glycines and poly-beta-alanines, respectively. Due to the substituted nitrogen atoms, the ability for hydrogen bond formation between peptide bonds gets lost. It may be very interesting to see whether such non-natural oligomers could be regarded as foldamers, which fold into definite backbone conformers. In this paper, we provide a complete overview on helix formation in alpha- and beta-peptoids on the basis of systematic theoretical conformational analyses employing the methods of ab initio molecular orbital (MO) theory. It can be shown that the alpha- and beta-peptoid structures form helical structures with both trans and cis peptide bonds despite the missing hydrogen bonds. Obviously, the conformational properties of the backbone are more important for folding than the possibility of hydrogen bonding. There are close relationships between the helices of alpha-peptoids and poly-glycine and poly-proline helices of alpha-peptides, whereas the helices of beta-peptoids correspond to the well-known helical structures of beta-peptides as, for instance, the 3(1)-helix of beta-peptides with 14-membered hydrogen-bonded rings. Thus, alpha- and beta-peptoids enrich the field of foldamers and may be used as useful tools in peptide and protein design.     

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.     

Expanded turn conformations: characterization and sequence-structure correspondence in alpha-turns with implications in helix folding

Like the beta-turns, which are characterized by a limiting distance between residues two positions apart (i, i+3), a distance criterion (involving residues at positions i and i+4) is used here to identify alpha-turns from a database of known protein structures. At least 15 classes of alpha-turns have been enumerated based on the location in the phi,psi space of the three central residues (i+1 to i+3)-one of the major being the class AAA, where the residues occupy the conventional helical backbone torsion angles. However, moving towards the C-terminal end of the turn, there is a shift in the phi,psi angles towards more negative phi, such that the electrostatic repulsion between two consecutive carbonyl oxygen atoms is reduced. Except for the last position (i+4), there is not much similarity in residue composition at different positions of hydrogen and non-hydrogen bonded AAA turns. The presence or absence of Pro at i+1 position of alpha- and beta-turns has a bearing on whether the turn is hydrogen-bonded or without a hydrogen bond. In the tertiary structure, alpha-turns are more likely to be found in beta-hairpin loops. The residue composition at the beginning of the hydrogen bonded AAA alpha-turn has similarity with type I beta-turn and N-terminal positions of helices, but the last position matches with the C-terminal capping position of helices, suggesting that the existence of a "helix cap signal" at i+4 position prevents alpha-turns from growing into helices. Our results also provide new insights into alpha-helix nucleation and folding.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

An automated method for consistent helix assignment using turn information

A new automated helix assignment method is presented that leads to a more consistent definition of the helix termini, especially of the helix C-terminus. The method assigns a helix to segments of protein chain where adjacent helical turn structures are observed, capped by specific distorted turn types (e.g., open helical turns without a hydrogen bond) or capping motifs (e.g., the Schellman motif). Helix termini are detected by observing the behavior of the NH group in N-termini and the CO group in C-termini; in each case, the respective group must be free to interact with hydrogen bonding partners outside of the putative helix for a helix terminus to be assigned. The presented assignment method and SHAFT-assigned helices are part of Secbase and are made available with Relibase+ 3.0 and the free web version of Relibase 3.0. The method can also be used for the helix assignments of additional protein structures.     

Combined use of molecular dynamics simulations and NMR to explore peptide bond isomerization and multiple intramolecular hydrogen-bonding possibilities in a cyclic pentapeptide, cyclo(Gly-Pro-D-Phe-Gly-Val).

The conformational behavior of a model cyclic pentapeptide--cyclo(Gly-L-Pro-D-Phe-Gly-L-Val)--has been explored through the combined use of in vacuo molecular dynamics simulations and a range of nmr experiments (preceding paper). The molecular dynamics analysis suggests that, despite the conformational constraints imposed by formation of the pentapeptide cycle, this pentapeptide undergoes conformational transitions between various hydrogen-bonded conformations, characterized by low energy barriers. An inverse gamma turn with Pro in position i + 1 and a gamma turn with D-Phe in position i + 1 are two alternatives occurring frequently. Like other DLDDL cyclic pentapeptides, cyclo(Gly-Pro-D-Phe-Gly-Val) is also stabilized by an inverse gamma-turn structure with the beta-branched Val residue in position i + 1, and this hydrogen bond is retained in the different conformational families. The gamma-turn around D-Phe3 and the inverse gamma turn around Val5 are consistent with the nmr observations. 3JNH-CH alpha coupling constants of the all-trans forms were calculated from one of the molecular dynamics trajectories and are comparable to nmr experimental data, suggesting that the conformational states visited during the simulation are representative of the conformational distribution in solution. In addition to the equilibrium among various hydrogen-bonded all-trans conformers, the observation in nmr spectra of two sets of resonances for all peptide protons indicated a slow conformational interconversion of the Gly-Pro peptide bond between trans and cis isomers. The activation energy between these two conformers was determined experimentally by magnetization transfer and was calculated by high temperature constrained molecular dynamics simulation. Both methods yield a free energy of activation of ca. 20 kcal/mol. Furthermore, the free energy of activation is dependent on the direction of rotation of the Gly-Pro peptide bond.     

Importance of context in protein folding: secondary structural propensities versus tertiary contact-assisted secondary structure formation

Molecular dynamics simulations can be used to reveal the detailed conformational behaviors of peptides and proteins. By comparing fragment and full-length protein simulations, we can investigate the role of each peptide segment in the folding process. Here, we take advantage of information regarding the helix formation process from our previous simulations of barnase and protein A as well as new simulations of four helical fragments from these proteins at three different temperatures, starting with both helical and extended structures. Segments with high helical propensity began the folding process by tethering the chain through side chain interactions involving either polar interactions, such as salt bridges, or hydrophobic staples. These tethers were frequently nonnative (i.e., not i --> i + 4 spacing) and provided a scaffold for other residues, thereby limiting the conformational search. The helical structure then propagated on both sides of the tether. Segments with low stability and propensity formed later in the folding process and utilized contacts with other portions of the protein when folding. These helices formed via a tertiary contact-assisted mechanism, primarily via hydrophobic contacts between residues distant in sequence. Thus, segments with different helical propensities appear to play different roles during protein folding. Furthermore, the active role of nonlocal side chains in helix formation highlights why we must move beyond simple hierarchical models of protein folding.     

beta-hairpin stability and folding: molecular dynamics studies of the first beta-hairpin of tendamistat

The stability and (un)folding of the 19-residue peptide, SCVTLYQSWRYSQADNGCA, corresponding to the first beta-hairpin (residues 10 to 28) of the alpha-amylase inhibitor tendamistat (PDB entry 3AIT) has been studied by molecular dynamics simulations in explicit water under periodic boundary conditions at several temperatures (300 K, 360 K and 400 K), starting from various conformations for simulation lengths, ranging from 10 to 30 ns. Comparison of trajectories of the reduced and oxidized native peptides reveals the importance of the disulphide bridge closing the beta-hairpin in maintaining a proper turn conformation, thereby insuring a proper side-chain arrangement of the conserved turn residues. This allows rationalization of the conservation of those cysteine residues among the family of alpha-amylase inhibitors. High temperature simulations starting from widely different initial configurations (native beta-hairpin, alpha and left-handed helical and extended conformations) begin sampling similar regions of the conformational space within tens of nanoseconds, and both native and non-native beta-hairpin conformations are recovered. Transitions between conformational clusters are accompanied by an increase in energy fluctuations, which is consistent with the increase in heat capacity measured experimentally upon protein folding. The folding events observed in the various simulations support a model for beta-hairpin formation in which the turn is formed first, followed by hydrogen bond formation closing the hairpin, and subsequent stabilization by side-chain hydrophobic interactions.     

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.     

Turns revisited: a uniform and comprehensive classification of normal, open, and reverse turn families minimizing unassigned random chain portions

Turns are irregular secondary structure elements with a hydrogen bond or a specific Calpha-Calpha distance between the first and the last residue. They are up to six residues in length. Here, we present a uniform classification for all normal (CO(i) - NH(i+n) hydrogen bond), open (a Calpha(i)-Calpha(i+n) distance up to 10 A), and reverse (NH(i) - CO(i+i) hydrogen bond) turn families based on current structural data. Considering the large amount of data evaluated, this classification likely covers quite comprehensively most of the possible conformations of turns. All turn structures of a nonredundant dataset of 1903 protein chains were retrieved using Relibase and clustered using emergent self-organizing maps. This leads to three normal, four open, and five reverse turn families with several new turn-types. Based on the amino acid propensities, the achieved separation into normal, open, and reverse turn families seems convincing. In combination with beta-sheet and helix classification on average 96% of the given protein chain can now be successfully classified.     

Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.     

Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.     

A C-H triplebond O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins

The serendipitous observation of a C-H cdots, three dots, centered O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H triplebond O interaction between the T-4 C(alpha)H and T+1 Cz doublebond O group (C triplebond O< or =3.5A) becomes possible only when the T+1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T-4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T-4 position with the motif being further stabilized by the formation of a side-chain-backbone O triplebond H-N hydrogen bond involving the side-chain of residue T-4 and the N-H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H triplebond O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H cdots, three dots, centered O hydrogen bonds between (T-4) C(alpha)H triplebond O (T+1) and (T-8) C(alpha)H triplebondC doublebond O (T+3).