Conformational preferences of proline analogues with different ring size

The conformational study on L-azetidine-2-carboxylic acid (Ac-Aze-NHMe, the Aze dipeptide) and (S)-piperidine-2-carboxylic acid (Ac-Pip-NHMe, the Pip dipeptide) is carried out using ab initio HF and density functional methods with the self-consistent reaction field method to explore the differences in conformational preferences and cis-trans isomerization for proline residue and its analogues with different ring size in the gas phase and in solution (chloroform and water). The change of ring size by deleting a CH2 group from or adding a CH2 group to the prolyl ring results the remarkable changes in backbone and ring structures compared with those of the Pro dipeptide, especially in the C'-N imide bond length and the bond angles around the N-C(alpha) bond. The four-membered azetidine ring can have either puckered structure depending on the backbone structure because of the less puckered structure. The six-membered piperidine ring can adopt chair and boat conformations, but the chair conformation is more preferred than the boat conformation. These calculated preferences for puckering are consistent with experimental results from analysis of X-ray structures of Aze- and Pip-containing peptides. On going from Pro to Aze to Pip, the axiality (i.e., a tendency to adopt the axial orientation) of the NHMe group becomes stronger, which can be ascribed to reduce the steric hindrances between 1,2-substituted Ac and NHMe groups. As the solvent polarity increases, the polyproline II-like conformation becomes more populated and the relative stability of conformation tC with a C7 hydrogen bond between C'=O of the amino group and N-H of the carboxyl group decreases for both the Aze and Pip dipeptides, as seen for the Pro dipeptide. The cis population and rotational barriers for the imide bond increase with the increase of solvent polarity for both the Aze and Pip dipeptides, as seen for the Pro dipeptide. In particular, the cis-trans isomerization proceeds in common through only the clockwise rotation with omega' approximately +120 degrees about azetyl and piperidyl peptide bonds in the gas phase and in solution, as seen for alanyl and prolyl peptide bonds. The pertinent distance d(N...H-N(NHMe)) and the pyramidality of imide nitrogen can describe the role of this hydrogen bond in stabilizing the transition state structure, but the lower rotational barriers for the Aze and Pip dipeptides than those for the Pro dipeptide, which is observed from experiments, cannot be rationalized.     

Investigations of the Mechanism of the “Proline Effect” in Tandem Mass Spectrometry Experiments: The “Pipecolic Acid Effect”

The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established “proline effect” through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give y_n ⁺ ions. In contrast, Pip- and NMA-fragment through a different mechanism, the “pipecolic acid effect,” selectively at the amide bond C-terminal to the Pip/NMA residue to give b_n ⁺ ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the “proline effect’ can in part be rationalized by the increased basicity of the prolyl-amide site, the “pipecolic acid effect” cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the “pipecolic acid effect” is stronger than the “proline effect.” Figure

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Synthesis and conformational analysis of small peptides containing 6-endo-BT(t)L scaffolds as reverse turn mimetics

Two new dipeptide isosteres derived from L-leucine and meso-tartaric acid derivatives, named 6-endo-BTL and 6-endo-BtL, were inserted in a small peptide by means of SPPS, and the conformational features of the resulting peptides 3 and 4 were studied by NMR, IR, and molecular modeling techniques. The presence of a reverse turn conformation was observed in all the structures, suggesting the key role of the scaffolds as reverse turn promoters. Peptides 3 and 4 did not adopt a preferred conformation as indicated by the presence of equilibria between open turn and intramolecular hydrogen-bonded structures. 6-endo-BTL-peptide 3 showed a 3:1 mixture of conformers. The major conformer adopted mainly an open turn structure in equilibrium with hydrogen-bonded structures. The minor conformer displayed a better organized structure with a 14-membered ring hydrogen-bond typical of a beta-hairpin-like structure, in equilibrium with a gamma-turn, too. 6-endo-BtL-peptide 4 showed a unique conformer, and did not adopt as good a conformation as 3, due to the bulky equatorial substituent at C-2. Thus, marked structural differences between peptides containing 6-endo-BTL and 6-endo-BtL scaffolds as reverse turn inducers exist.     

A reverse turn structure induced by a D,L-alpha-aminoxy acid dimer

Our previous work revealed that two adjacent D-alpha-aminoxy acids could form two homochiral N-O turns, with the backbone folding into an extended helical structure (1.8(8)-helix). Here, we report the conformational studies of linear peptides 3-6, which contain a D,L-alpha-aminoxy acid dimer segment. The NMR and X-ray analysis of 3 showed that it folded into a loop conformation with two heterochiral N-O turns. This loop segment can be used to constrain tetrapeptides 4 and 6 to form a reverse turn structure. (1)H NMR dilution studies, DMSO-d6 addition studies, and 2D-NOESY data indicated that tetrapeptides 4 and 6 folded into reverse turn conformations featured by a head-to-tail 16-membered-ring intramolecular hydrogen bond. In contrast, tetrapeptide 5 with L-Ala instead of Gly or D-Ala as the N-terminal amino acid could not form the desired reverse turn structure for steric reasons. Quantum mechanics calculations showed that model pentamide 7, with the same substitution pattern of 4, adopted a novel reverse turn conformation featuring two heterochiral N-O turns (each of an 8-membered ring hydrogen bond), a cross-strand 16-membered ring hydrogen bond, and a 7-membered ring gamma-turn.     

Stereoelectronic tuning of the structure and stability of the trp cage miniprotein

Proline residues are critical structural elements in proteins, defining turns, loops, secondary structure boundaries, and polyproline helices. Control of proline conformation therefore may be used to define protein structure and stability. 4-Substituted proline derivatives may be used to control proline ring pucker, which correlates with protein main chain conformation. To examine the use of proline conformational restriction to tune globular protein stability, a series of peptides derived from the trp cage miniprotein was synthesized. Proline at residue 12 of the trp cage miniprotein, which adopts a Cgamma-exo ring pucker in the NMR structure, was replaced with 4-substituted proline derivatives, including 4R derivatives favoring a Cgamma-exo ring pucker and 4S derivatives favoring a Cgamma-endo ring pucker. Eight trp cage peptides were synthesized, five of which included residues that are not commercially available, without requiring any solution phase chemistry. Analysis of the trp cage peptides by circular dichroism and NMR indicated that the structure and stability of the trp cage miniprotein was controllable based on the conformational bias of the proline derivative. Replacement of Pro12 with 4S-substituted proline derivatives that favor the Cgamma-endo ring pucker destabilized the trp cage, while replacement of Pro12 with 4R-substituted proline derivatives that favor a Cgamma-exo ring pucker resulted in increased alpha-helicity and thermal stability of the trp cage. The most stable trp cage derivatives contained benzoates of 4R-hydroxyproline, which also exhibited the most pronounced stereoelectronic effects in TYProxN model peptides. Overall, the stability of the trp cage was tunable by over 50 degrees C depending on the identity of the proline side chain at residue 12.     

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.     

Conformational energy calculation on the neurotensin c-terminal pentapeptide Arg–Pro–Tyr–Ile–Leu OH

The conformational behavior of the C-terminal neurotensin pentapeptide, Arg–Pro–Tyr–Ile–Leu OH [NT(9–13)], was investigated using empirical energy calculations. A special aim was to display the specific contribution of each residue to induce conformations able to interact with biological receptors. Restrictions were then introduced in intramolecular interactions involving the Arg side chain and the terminal COOH group. The stablest conformations include in the order of decreasing stability: a distorted helical form for the C-terminal tetrapeptide, a (Pro₂–Tyr₃) β turn I, an α helix, an extended form, and a (Tyr₂–Ile₃) β turn III, which are energetically rather close (ΔE < 3 kcal/mol). The NT(9–13) peptide appears then as a rather flexible molcule with a noteworthy ability of adaptation to a substrate. Extended forms would be in agreement with a zipper model of interactions with receptors, whereas folded forms involving helices and β, γ turns would support a lock and key model. The specific contribution of side chains, specially those of Tyr and Arg residues as well as the key position of the Pro residue emerge clearly from this study.     

Peptide backbone folding induced by the C(alpha)-tetrasubstituted cyclic alpha-amino acids 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) and 1-aminocyclopentane-1-carboxylic acid (Ac5c). A joint computational and experimental study

The conformational study of a new group of synthetic peptides containing 4-amino-1,2-dithiolane-4-carboxylic acid (Adt), a cysteine-related achiral residue, has been carried out through a joint application of computational and experimental methodologies. Molecular Dynamics simulations clearly suggest the tendency of this molecule to adopt a gamma-turn conformation in vacuum and help in analyzing the complex and crucial conformational behaviour of the dithiolane ring which appears to preferentially adopt a C(S)-like structure. Electronic structure calculations carried out in solution using the Density Functional Theory also indicate the preservation of the gamma-like folding in apolar solvents and the helix-like one in more polar solvents. A comparison with the achiral 1-aminocycloalkane-1-carboxylic acid (Ac5c) has been carried out using the same computational tools. NMR and IR data on dipeptide derivatives containing the Adt or Ac5c residue show that in chloroform solution all the models prefer a gamma-turn structure, centered at the cyclic residue, stabilized by an intramolecular H-bond, whereas in a more polar solvent, i.e. dimethyl sulfoxide, this folding is not maintained. The experimental conformational studies, extended to N-Boc protected tripeptides, clearly indicate the remarkable tendency of both the five-membered C(alpha)-tetrasubstituted cyclic amino acids Adt and Ac5c to induce the gamma-turn structure also in models able to adopt the beta-bend conformation.     

A Multicomponent Conjugation Strategy to Unique N‐Steroidal Peptides: First Evidence of the Steroidal Nucleus as a β‐Turn Inducer in Acyclic Peptides

Constraining small peptides into specific secondary structures has been a major challenge in peptide ligand design. So far, the major solution for decreasing the conformational flexibility in small peptides has been cyclization. An alternative is the use of topological templates, which are able to induce and/or stabilize peptide secondary structures by means of covalent attachment to the peptide. Herein a multicomponent strategy and structural analysis of a new type of peptidosteroid architecture having the steroid as N‐substituent of an internal amide bond is reported. The approach comprises the one‐pot conjugation of two peptide chains (or amino acid derivatives) to aminosteroids by means of the Ugi reaction to give a unique family of N‐steroidal peptides. The conjugation efficiency of a variety of peptide sequences and steroidal amines, as well as their consecutive head‐to‐tail cyclization to produce chimeric cyclopeptide–steroid conjugates, that is, macrocyclic lipopeptides, was assessed. Determination of the three‐dimensional structure of an acyclic N‐steroidal peptide in solution proved that the bulky, rigid steroidal template is capable of both increasing significantly the conformational rigidity, even in a peptide sequence as short as five amino acid residues, and inducing a β‐turn secondary structure even in the all‐s‐trans isomer. This report provides the first evidence of the steroid skeleton as β‐turn inducer in linear peptide sequences.     

Conformational profile of a proline-arginine hybrid

The intrinsic conformational preferences of a new nonproteinogenic amino acid have been explored by computational methods. This tailored molecule, named ((β)Pro)Arg, is conceived as a replacement for arginine in bioactive peptides when the stabilization of folded turn-like conformations is required. The new residue features a proline skeleton that bears the guanidilated side chain of arginine at the C(β) position of the five-membered pyrrolidine ring, in either a cis or a trans orientation with respect to the carboxylic acid. The conformational profiles of the N-acetyl-N'-methylamide derivatives of the cis and trans isomers of ((β)Pro)Arg have been examined in the gas phase and in solution by B3LYP/6-31+G(d,p) calculations and molecular dynamics simulations. The main conformational features of both isomers represent a balance between geometric restrictions imposed by the five-membered pyrrolidine ring and the ability of the guanidilated side chain to interact with the backbone through hydrogen bonds. Thus, both cis- and trans-((β)Pro)Arg exhibit a preference for the α(L) conformation as a consequence of the interactions established between the guanidinium moiety and the main-chain amide groups.     

The special five-membered ring of proline: An experimental and theoretical investigation of alkali metal cation interactions with proline and its four- and six-membered ring analogues

The interaction of the alkali metal cations, Li+, Na+, and K+, with the amino acid proline (Pro) and its four- and six-membered ring analogues, azetidine-2-carboxylic acid (Aze) and pipecolic acid (Pip), are examined in detail. Experimentally, threshold collision-induced dissociation of the M+(L) complexes, where M = Li, Na, and K and L = Pro, Aze, and Pip, with Xe are studied using a guided ion beam tandem mass spectrometer. From analysis of the kinetic energy dependent cross sections, M(+)-L bond dissociation energies are measured. These analyses account for unimolecular decay rates, internal energy of reactant ions, and multiple ion-molecule collisions. Ab initio calculations for a number of geometric conformations of the M+(L) complexes were determined at the B3LYP/6-311G(d,p) level with single-point energies calculated at MP2(full), B3LYP, and B3P86 levels using a 6-311+G(2d,2p) basis set. Theoretical bond energies show good agreement with the experimental bond energies, which establishes that the zwitterionic form of the alkali metal cation/amino acid, the lowest energy conformation, is formed in all cases. Despite the increased conformational mobility in the Pip systems, the Li+, Na+, and K+ complexes of Pro show higher binding energies. A meticulous examination of the zwitterionic structures of these complexes provides an explanation for the stability of the five-membered ring complexes.     

Conformational analysis and mu-opioid receptor affinity of short peptides, endomorphin models in a low polarity solvent

Peptide carbamates containing the sequence H-Pro-Trp-PheNH2 showed in CDCl3 restricted conformations stabilized by the presence of a gamma-turn. To test the reliability of the peptides as endomorphin conformational models, we measured the affinities for mu-receptors labelled with [3H]-DAMGO. In particular, Cbz-Pro-Trp-PheNH2 displayed a nanomolar affinity.     

Conformational preferences of pseudoproline residues

The conformational study on N-acetyl-N'-methylamides of oxazolidine and thiazolidine residues (Ac-Oxa-NHMe and Ac-Thz-NHMe) is carried out using ab initio HF and density functional B3LYP methods with the self-consistent reaction field method to explore the effects of the replacement of the C(gamma)H(2) group in the prolyl ring by oxygen or sulfur atoms on the conformational preferences and prolyl cis-trans isomerization in the gas phase and in solution (chloroform and water). As the solvent polarity increases, the conformations C with the C7 intramolecular hydrogen bonds become depopulated, the PPII- or PPI-like conformations F become more populated, and the cis populations increase for both Oxa and Thz dipeptides, as found for the Pro dipeptide, although the populations of backbone conformations and puckerings are different in pseudoproline and proline dipeptides. As the increase of solvent polarity, the populations of the trans/up conformations decrease for Oxa and Thz dipeptides, but they increase for the Pro dipeptide. It is found that the cis-trans isomerization proceeds through the anticlockwise rotation with omega' approximately -60 degrees about the oxazolidyl peptide bond and the clockwise rotation with omega' approximately +120 degrees about the thiazolidyl peptide bond in the gas phase and in solution, whereas the clockwise rotation is preferred for the prolyl peptide bond. The pertinent distance d(N...H-N(NHMe)) and the pyramidality of the prolyl nitrogen can describe the role of this hydrogen bond in stabilizing the transition state structure but the lower rotational barriers for Oxa and Thz dipeptides than those for the Pro dipeptide, which is observed from experiments, cannot be rationalized. The calculated cis populations and rotational barriers to the cis-trans isomerization for both Oxa and Thz dipeptides in chloroform and/or water are consistent with the experimental values.     

Conformational behavior of peptides containing residues of 3-azetidinesulfonic (3AzeS) and 4-piperidinemethanesulfonic (4PiMS) acids

The conformational behavior of four model peptides containing residues of 3-azetidinesulfonic (3AzeS) and 4-piperidinemethane sulfonic (4PiMS) acids was studied in both a crystalline state and in solution using X-ray, NMR, and IR experiments. It was found that in the crystalline state, both of the models di- and tripeptides studied adopted extended conformations and demonstrated considerable conformational flexibility. In solution, it is likely that a flexible ensemble of conformations is adopted, including extended structures and more compact ones without persistent hydrogen bonds. One of the most interesting features of the peptides was the axial chirality observed due to the slow rotation around the amide bond formed by the endocyclic nitrogen atoms of the non-chiral 3AzeS and 4PiMS residues. It was shown for one of the derivatives that the configuration of the chiral axis had an impact on the conformation of the neighboring amino acid residue.     

Conformational manifold of alpha-aminoisobutyric acid (Aib) containing alanine-based tripeptides in aqueous solution explored by vibrational spectroscopy, electronic circular dichroism spectroscopy, and molecular dynamics simulations

Replacement of the alpha-proton of an alanine residue to generate alpha-aminoisobutyric acid (Aib) in alanine-based oligopeptides favors the formation of a 3(10) helix when the length of the oligopeptide is about four to six residues. This research was aimed at experimentally identifying the structural impact of an individual Aib residue in an alanine context of short peptides in water and Aib's influence on the conformation of nearest-neighbor residues. The amide I band profile of the IR, isotropic and anisotropic Raman, and vibrational circular dichroism (VCD) spectra of Ac-Ala-Ala-Aib-OMe, Ac-Ala-Aib-Ala-OMe, and Ac-Aib-Ala-Ala-OMe were measured and analyzed in terms of different structural models by utilizing an algorithm that exploits the excitonic coupling between amide I' modes. The conformational search was guided by the respective 1H NMR and electronic circular dichroism spectra of the respective peptides, which were also recorded. From these analyses, all peptides adopted multiple conformations. Aib predominantly sampled the right-handed and left-handed 3(10)-helix region and to a minor extent the bridge region between the polyproline (PPII) and the helical regions of the Ramachandran plot. Generally, alanine showed the anticipated PPII propensity, but its conformational equilibrium was shifted towards helical conformations in Ac-Aib-Ala-Ala-OMe, indicating that Aib can induce helical conformations of neighboring residues positioned towards the C-terminal direction of the peptide. An energy landscape exploration by molecular dynamics simulations corroborated the results of the spectroscopic studies. They also revealed the dynamics and pathways of potential conformational transitions of the corresponding Aib residues.     

Beta-hairpin folding by a model amyloid peptide in solution and at an interface

The development of specific agents against amyloidoses requires an understanding of the conformational distribution of fibrillogenic peptides at a microscopic level. Here, I present molecular dynamics simulations of the model amyloid peptide LSFD with sequence LSFDNSGAITIG-NH2 in explicit water and at a water/vapor interface for a total time scale of approximately 1.8 micros. An extended structure was used as initial peptide configuration. At approximately 290 K, solvated LSFD was kinetically trapped in diverse misfolded beta-sheet/coil conformations. At 350 K, in contrast, the same type II' beta-hairpin in equilibrium with less ordered but also U-shaped conformations was observed for the core residues DNSGAITI in solution and at the interface in multiple independent simulations. The most stable structural unit of the beta-hairpin was the two residue turn (GA). The core residues exhibited a well-defined folded state in which the beta-hairpin was stabilized by a hydrogen bond between the side chain of Asn-385 and the main chain carbonyl group of Gly-387. My results suggest that beta-sheet conformations indicated from previous Fourier-transform infrared spectroscopy measurements immediately after preparation of the peptide solution may not arise from protofilaments as speculated by others but are a property of LSFD monomers. In addition, combined with previous results from X-ray scattering, my findings suggest that interfacial aggregation of LSFD implies a transition from U-shaped to extended peptide conformations. This work including the first simulations of reversible beta-hairpin folding at an interface is an essential step toward a microscopic understanding of interfacial peptide folding and self-assembly. Knowledge of the main conformation of the peptide core may facilitate the design of possible inhibitors of LSFD aggregation as a test ground for future computational therapeutic strategies against amyloid diseases.     

Molecular Level Characterization of the Structure and Interactions in Peptide‐Functionalized Metal–Organic Frameworks

We use density functional theory, newly parameterized molecular dynamics simulations, and last generation ¹⁵N dynamic nuclear polarization surface enhanced solid‐state NMR spectroscopy (DNP SENS) to understand graft–host interactions and effects imposed by the metal–organic framework (MOF) host on peptide conformations in a peptide‐functionalized MOF. Focusing on two grafts typified by MIL‐68‐proline ( ‐Pro ) and MIL‐68‐glycine‐proline ( ‐Gly‐Pro ), we identified the most likely peptide conformations adopted in the functionalized hybrid frameworks. We found that hydrogen bond interactions between the graft and the surface hydroxyl groups of the MOF are essential in determining the peptides conformation(s). DNP SENS methodology shows unprecedented signal enhancements when applied to these peptide‐functionalized MOFs. The calculated chemical shifts of selected MIL‐68‐NH‐ Pro and MIL‐68‐NH‐ Gly‐Pro conformations are in a good agreement with the experimentally obtained ¹⁵N NMR signals. The study shows that the conformations of peptides when grafted in a MOF host are unlikely to be freely distributed, and conformational selection is directed by strong host–guest interactions.     

寡肽Asterin B和C溶液构象的NMR研究1: ^1H NMR归属及构象特征

Asterin B和C是从紫菀中分得的两个寡肽, 本文利用2D-NMR技术归属了它们的^1H NMR谱线, 并讨论了它们的构象特征。为进一步采用NMR和分子动力学(MD)方法研究它们的溶液构象奠定了基础。     

Effect of 3-hydroxyproline residues on collagen stability

Collagen is an integral part of many types of connective tissue in animals, especially skin, bones, cartilage, and basement membranes. A fibrous protein, collagen has a triple-helical structure, which is comprised of strands with a repeating Xaa-Yaa-Gly sequence. l-Proline (Pro) and 4(R)-hydroxy-l-proline (4-Hyp) residues occur most often in the Xaa and Yaa positions. The 4-Hyp residue is known to increase markedly the conformational stability of a collagen triple helix. In natural collagen, a 3(S)-hydroxy-l-proline (3-Hyp) residue occurs in the sequence: 3-Hyp-4-Hyp-Gly. Its effect on collagen stability is unknown. Here, two host-guest peptides containing 3-Hyp are synthesized: (Pro-4-Hyp-Gly)(3)-3-Hyp-4-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 1) and (Pro-4-Hyp-Gly)(3)-Pro-3-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 2). The 3-Hyp residues in these two peptides diminish triple-helical stability in comparison to Pro. This destabilization is small when 3-Hyp is in the natural Xaa position (peptide 1). There, the inductive effect of its 3-hydroxyl group diminishes slightly the strength of the interstrand 3-HypC=O.H-NGly hydrogen bond. The destabilization is large when 3-Hyp is in the nonnatural Yaa position (peptide 2). There, its pyrrolidine ring pucker leads to inappropriate mainchain dihedral angles and interstrand steric clashes. Thus, the natural regioisomeric residues 3-Hyp and 4-Hyp have distinct effects on the conformational stability of the collagen triple helix.