Proton transfer in and polarizability of hydrogen bonds coupled with conformational changes in proteins. II. IR investigation of polyhistidine with various carboxylic acids

Polyhistidine-carboxylic acid systems are studied by ir spectroscopy. It is shown that OH ?N ? O^?…H⁺N bonds formed between carboxylic groups and histidine residues are easily polarizable proton-transfer hydrogen bonds when the pK_a of the protonated histidine residues is about 2.8 units larger than that of the carboxylic groups. From these results it bis concluded that OH ?N ? O^? ?H⁺N bonds between glutamic or aspartic acid histidine residues in proteins may be easily polarizable proton-transfer bonds. Furthermore, it is demonstrated that water molecules shift the proton-transfer equilibria in these hydrogen bonds in favor of the polar structure, i.e., due to water or polar environments OH ?N ? O^? ?H⁺N bonds with smaller ΔpK_a values become easily polarizable proton-transfer hydrogen bonds. A consideration of the amide bands of polyhistidine shows that it can be present in five different conformations. It is shown that these conformational changes are strongly related to the degree of proton transfer. Hence, the degree of proton transfer, the degree of hydration, and conformation are not independent of each other, but are strongly coupled. Further proof for the interdependence of proton transfer and conformational changes are hysteresis effects, which are observed with studies of polyhistidine dependent on carboxylic acid, adsorption and desorption. OH ?N ? O^? ?H⁺N bonds between aspartic and glutamic acid and histidine residues are present in hemoglobin, in ribonucleases, and in proteases, whereby this type of bond is preferentially found in the active centers of these enzymes. It is pointed out that hydrogen bonds with such interaction properties should be of great significance for structure and especially functions of proteins in which they are present.     

Packing analysis of carbohydrates and polysaccharides. IV. A new method for detailed crystal structure refinement of polysaccharides and its application to V-amylose

A method is described for predicting and solving crystal structures of linear homopolysaccharides. The method is based on the refinement of the structure with respect to either stereochemical constraints or x-ray diffraction intensities. In the refinement process, all conformational and packing features of the molecule, such as bond lengths, bond angles, conformational angles, nonbonded contacts, hydrogen bonds, etc., can be allowed to vary until the structure reaches both a conformation and crystalline packing that are in minimum disagreement with the stereochemical restraints and the diffraction data. In this fashion, both packing and conformational features of the structure can be simultaneously refined, and not separately as has been the custom in the past. The refinement procedure is based on a method of constrained optimization which possesses improved characteristics of reaching a solution and avoiding false minima, in comparison with least squares methods. The procedure is, in addition, capable of easily finding molecules of solvent of crystallization. The method was applied to further refining the previously solved crystal structure of V-amylose. The results indicated that contrary to the previously found six-fold molecular symmetry in the P2₁2₁2₁ space group, the V-amylose molecule exhibits only two-fold symmetry with the asymmetric unit consisting of three glucose residues in one-half turn of the helix. The three residues are nonequivalent principally due to unequal rotational positions of the hydroxymethyl groups. The crystal structure of V-amylose predicted from stereochemical refinement was identical in all details with that obtained from refining against X-ray data. The excellent agreement with the diffraction data was indicated by the crystallographic disagreement index R = 0.25.     

Two-H-Bonded and One-H-Bonded Structure Alternations in Collagen

Abstract

This paper concerns the conformational variability of collagen as related to the concrete tripeptides (GXY)_n constituting its primary structure. The previously elaborated model (V.G. Tumanyan, N.G. Esipova, Biophysics 28, 1021–1025, 1983) with two nets of hydrogen bonds is useful for tripeptides where X is an amino acid. If X is an imino acid, the common one-bonded Rich & Crick model is valid. In this work, compound sequences including tripeptides of different types are considered. Molecular mechanics is used to assess the conformations of the junction regions when a structure with two nets of hydrogen bonds precedes the structure with one net, and vice versa. Thus, all types of sequences typical for natural collagen are covered. It is shown that the combined model representing an alternation of the two-H-bonded model and the one-H-bonded Rich & Crick model is satisfactory stere-ochemically, and provides more favorable energy in comparison with the continuous one-H-bonded model. Besides, a more favorable hydration of the molecule occurs in this case. Some conclusions are made about interchain and intrachain ionic bonds. Thus, it is deduced for the concrete fibrillar protein how a one-dimensional structure determines three-dimensional structure. The macromolecular structure thus suggested is in accord with the experimental data on hydrogen exchange.     

Quantum-mechanical kinetic study of the primary reaction of the photochemical cycle of Halobacterium halobium

A two-frequency oscillator model for the primary photochemical reaction bacteriorhodopsin batho-bacteriorhodopsin (K₆₁₀) is proposed. According to this model two conformational changes in the reaction are considered to take place: the first one is a distortion of the retinal in the bacteriorhodopsin active site and the second one is a conformational transition of the bacterioopsin, affecting the native structure hydrogen bonds. On the basis of this model the temperature dependences of the rate constants for normal and deuterated reactants are calculated in good agreement with the available experimental data. The relations of the reaction considered to the primary photochemical reaction of vision are discussed.     

Structurally symmetrical,easily polarizable hydrogen bonds between side chains in proteins and proton-conducting mechanisms. III

(L -Cys)_n, (L -Lys)_n, and (L -Glu)_n were studied by ir spectroscopy in terms of their degree of deprotonation or protonation. It is shown that structurally symmetrical, easily polarizable SH ?S^? ? ^?S ?HS, N⁺H ?N ? N ?H⁺N, and OH ?O^? ? ^?O ?HO hydrogen bonds are formed between the side chains. The different wave number distributions of the ir continua caused by these hydrogen bonds show that the barrier in the double-minimum proton potential decreases in the series of these hydrogen bonds. The stability of these hydrogen bonds against hydration increases in this series. The OH ?O^? ? ^?O ?HO bonds are not broken by small amounts of water. With (L -Cys)_n the formation of easily polarizable hydrogen bonds and a β-structure–coil transition are strongly interdependent. As a result of this coupling effect, the β-structure–coil transition becomes cooperative. With (L -Glu)_n, the formation of the polarizable hydrogen bonds and the observed conformational change are independent processes. The (L -Glu)_n conformation changes from α-helix to coil only if more than 80% of the residues are deprotonated. Finally, on the basis of the various types of easily polarizable hydrogen bonds, charge shifts in active centers of enzymes and the proton-conducting mechanism through hydrophobic regions of biological membranes are discussed.     

Possible formation of the left-handed alpha-helix of poly-L-serine

A conformational study of poly-L -serine has shown that it can exist in the left-handed α-helical form. A study of a pair of peptide units with the serine sidegroup attached to the α carbon atom linking the two units showed that O? H ?O hydrogen bonds between the OH group of the side chain and a carbonyl oxygen of the first peptide group in the backbone can occur in two regions of ?, namely, ? = 15°–30° for χ₁ = 300° and for ? = 225°-230° for ? = 60°. The latter is close to a possible left-handed helix of poly-L -serine, stabilized by N? H ?O hydrogen bonds. From a study of contact criteria, the best conformation for this helix is found to be ? = 227°, Ψ = 238°, χ₁ = 65° which has n = 3.65, h = 1.51 A. The N? H ?O hydrogen bond has a length of 2.90 A. (6°) and the O? H ?O hydrogen bond is of length 2.60 A. (0°). There are no other bad short contacts in the structure. The cylindrical coordinates of the atoms, as well as a perspective view of the structure arc given in this paper.     

Low-energy conformers of pamidronate and their intramolecular hydrogen bonds: a DFT and QTAIM study

Extensive DFT and ab initio calculations were performed to characterize the conformational space of pamidronate, a typical pharmaceutical for bone diseases. Mono-, di- and tri-protic states of molecule, relevant for physiological pH range, were investigated for both canonical and zwitterionic tautomers. Semiempirical PM6 method were used for prescreening of the single bond rotamers followed by geometry optimizations at the B3LYP/6-31++G(d,p) and B3LYP/6-311++G(d,p) levels. For numerous identified low energy conformers the final electronic energies were determined at the MP2/6-311++G(2df,2p) level and corrected for thermal effects at B3LYP level. Solvation effects were also considered via the COSMO and C-PCM implicit models. Reasonable agreement was found between bond lengths and angle values in comparison with X-ray crystal structures. Relative equilibrium populations of different conformers were determined from molecular partition functions and the role of electronic, vibrational and rotational degrees of freedom on the stability of conformers were analyzed. For no level of theory is a zwitterionic structure stable in the gas-phase while solvation makes them available depending on the protonation state. Geometrically identified intramolecular hydrogen bonds were analyzed by QTAIM approach. All conformers exhibit strong inter-phosphonate hydrogen bonds and in most of them the alkyl-amine side chain is folded on the P-C-P backbone for further hydrogen bond formation.

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Abundance and arrangement of single,double and bifurcated hydrogen bonds in protein α-helices: Statistical analysis of PDB-select

Statistics are collected and analyzed for the possibility of hydrogen bonding in the secondary structures of globular proteins, based on geometric criteria. Double and bifurcated bonds are considered as pairs of admissible H-bonds with two proton donors or two proton acceptors, respectively. Most of such bonds belong to peptide groups in α-helices, with O _i …N _{i + 3} nearly as frequent as O _i …N _{i + 4}; in contrast, most of the 3/10-helical segments are too short to have any. Alternating double and bifurcated bonds in α-helices form an apparently cooperative network structure. A typical α-helical segment perhaps carries two stretches of the H-bond network broken in the middle. The constituent H-bonds are nonlinear: the hydrogen atom is off the straight line connecting the proton donor and proton acceptor atoms. This deflection is larger for H _{i + 3} vs. bond line O _i −N _{i + 3} than for H _{i + 4} vs. O _i −N _{i + 4}, and though the two kinds of bond have about the same length (exceeding those typical of low-molecular compounds), O _i …N _{i + 4} must be stronger than O _i …N _{i + 3}. Double/bifurcated bonds are also not coplanar, i.e., hydrogen atoms are beyond the N…O…N (or O…N…O) plane. The text was submitted by the authors in English.     

Spectroscopy and molecular docking study on the interaction of daidzein and genistein with pepsin

The interaction of pepsin with daidzein (Dai) or genistein (Gen) was investigated using spectroscopic techniques under simulated physiological conditions. Dai and Gen can quench the fluorescence of pepsin and the quenching mechanism was a static process. The binding site number n and apparent binding constant K were measured at different temperatures. The thermodynamic parameters ΔΗ, ΔG and ΔS were calculated. The results indicated that van der Waals forces and hydrogen bond formation played major roles in the interaction of Dai or Gen with pepsin. The binding distance between pepsin and Dai or Gen was calculated according to energy transfer theory. The results of synchronous fluorescence spectra showed that the microenvironment and conformation of pepsin were changed. UV absorption and 3D fluorescence spectra showed that the binding interaction disturbed the microenvironment of amino acid residues and induced conformational changes in pepsin. Molecular docking results showed that Dai and Gen entered into the hydrophobic cavity of pepsin and two hydrogen bonds formed between Dai or Gen and pepsin. The results demonstrated that the interaction behavior between Dai and Gen with pepsin was slightly different, which denoted that the 5‐hydroxyl group of Gen, to a certain extent, had an effect on ligand binding to proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular insights of benzodipyrazole as CDK2 inhibitors: combined molecular docking,molecular dynamics,and 3D QSAR studies

Abstract

Benzodipyrazoles have been previously evaluated for their in vitro CDK2 inhibitory activity. In the current investigation, we identified a six-feature common pharmacophore model (AADDRR.33) which is predicted to be responsible for CDK2 inhibition. An efficient 3D QSAR (r^2?=?0.98 and q^2?=?0.82) model was also constructed by employing PLS regression analysis. From the molecular docking studies, we examined the binding patterns of compound 7aa with the target protein and also calculated the binding energy using MM-GBSA calculations. Three hydrogen bonds with Lys 33, Glu 81, and Leu 83 are conserved even after 1000?ps run in a molecular dynamics simulation. We identified the slight displacement in bond lengths and the conformational changes occurred during the dynamics. The results also elucidated the protein residue–ligand interaction fractions which clearly explained the involvement of non-H-bond interactions.     

13C- and 1H-nuclear magnetic resonance spectroscopy of permethylated disaccharides

The equilibrium composition of D-psicose in water, as determined from its ¹³C n.m.r. spectrum, is 7:2:5:5 α-furanose:β-furanose:α-pyranose:β-pyranose. These data, which are discussed in relation to the anomeric and ring-form equilibria of the other 2-hexuloses, are in general agreement with expectations based on conformational analysis. However, although the ¹³C chemical-shift pattern of the β-pyranose is closely consistent with the 1C(D) conformation predicted for this anomer, that of the α-pyranose is less readily reconciled with its predicted C1(D) conformation. Usually, carbon-13 nuclei of the furanose rings are substantially less shielded than those of their pyranose counterparts; for 2-hexulopyranoses in general, overall ¹³C shielding is close to that of those aldopyranoses expected to have similar conformational free-energies. Spectral data are also reported for several derivatives (glycosides, ethers, and selectively deuterated compounds) that were utilized in analysis of the D-psicose spectrum.     

Conformational changes due to vicinal glycosylation: The branched α-l-Rhap(1–2)[β-d-Galp(1–3)]-β-d-Glc1-OMe trisaccharide compared with its parent disaccharides*

Conformations of the α-l -Rhap(1-2)-β-d -Glc1-OMe and β-d -Galp(1-3)-β-d -Glc1-OMe disaccharides and the branched title trisaccharide were examined in DMSO-d₆ solution by ¹H-nmr. The distance mapping procedure was based on rotating frame nuclear Overhauser effect (NOE) constraints involving C- and O-linked protons, and hydrogen-bond constraints manifested by the splitting of the OH nmr signals for partially deuteriated samples. An “isotopomer-selected NOE” method for the unequivocal identification of mutually hydrogen-bonded hydroxyl groups was suggested. The length of hydrogen bonds thus detected is considered the only one motionally nonaveraged nmr-derived constraint. Molecular mechanics and molecular dynamics methods were used to model the conformational properties of the studied oligosaccharides. Complex conformational search, relying on a regular Φ,Ψ-grid based scanning of the conformational space of the selected glycosidic linkage, combined with simultaneous modeling of different allowed orientations of the pendant groups and the third, neighboring sugar residue, has been carried out. Energy minimizations were performed for each member of the Φ,Ψ grid generated set of conformations. Conformational clustering has been done to group the minimized conformations into families with similar values of glycosidic torsion angles. Several stable syn and anti conformations were found for the 1→2 and 1→3 bonds in the studied disaccharides. Vicinal glycosylation affected strongly the occupancy of conformational states in both branches of the title trisaccharide. The preferred conformational family of the trisaccharide (with average Φ,Ψ values of 38°, 17° for the 1→2 and 48°, 1° for the 1→3 bond, respectively) was shown by nmr to be stabilized by intramolecular hydrogen bonding between the nonbonded Rha and Gal residues. © 1998 John Wiley & Sons, Inc. Biopoly 46: 417–432, 1998     

A combined theoretical and spectroscopic study of 4,6-di-O-acetyl-2,3-dideoxy-d-erythro-hex-2-enopyranosyl sulfamide: a novel glycosyl carbonic anhydrase IX inhibitor

The novel 4,6-di-O-acetyl-2,3-dideoxy-d-erythro-hex-2-enopyranosyl sulfamide, which exhibits selectivity for inhibiting isoform IX of carbonic anhydrase as overexpressed in many tumors, has been investigated from a combined theoretical and spectroscopic point of view. The conformational study of the compound shows that the α-anomeric form is more stable than the β-anomeric form from a thermodynamic point of view after including solvent effects. This fact suggests that the synthesis reaction could take place mainly under thermodynamic control as the main experimental product is the α-anomeric form of the sulfamide. Calculated α/β ratio is about 95:5, in excellent agreement with experimental data. Optimized geometries of the α-anomeric form agree quite well with crystallographic data. The inclusion of a solvent has negligible effects on the conformations. A detailed analysis of some geometric parameters shed light into the conformational behavior of the sulfamide in terms of both exo- and endo-anomeric effects and antiperiplanar relationships. Natural bond orbital calculations confirm those findings. Several intramolecular hydrogen bonds, characterized through the Atoms-in-Molecules theory, were found in the stable conformers. They, however, seem to play no relevant role in determining the relative stability of α conformers with respect to the β ones. Calculated ¹H and ¹³C NMR chemical shifts support previous findings concerning configuration and conformation assignments of the title sulfamide. The IR spectrum of the compound is recorded and reported for the first time and the assignment of some of the most important bands is accomplished with the aid of calculated harmonic vibrational frequencies.     

Insights into a highly conserved network of hydrogen bonds in the agonist binding site of nicotinic acetylcholine receptors: A structural and theoretical study

Structural and theoretical studies on the geometrical features of a hydrogen‐bond network occurring in the binding site of nicotinic acetylcholine receptors (nAChRs) and composed of interconnected WxPD (Trp‐x‐Pro‐Asp) and SWyz (Ser‐Trp‐yz) sequences from loops A and B, respectively, have been carried out. Multiple sequence alignments using as template the sequence of the apoform of Aplysia californica acetylcholine binding protein (Ac‐AChBP) show the strict conservation of serine and tryptophan residues of the loop B SWyz sequence. Considering a sample of 19 high resolution AChBP structures, the strong conformational preferences of the key tryptophan residue has been pointing out, whatever the form, free or bounded, of AChBP. The geometry of the motif hydrogen‐bond network has been characterized through the analyses of seven distances. The robustness of the various hydrogen‐bond interactions is pointed out, the one involving the aspartate carboxylate group and the serine residue being the shortest of the network. The role of a cooperative effect involving a NH(His145)…OH (Ser142) hydrogen bond is highlighted. Density functional theory calculations on several simplified models based on the motif hydrogen‐bond network allow probing the importance of the various hydrogen‐bond interactions. The removal of the Ser142 hydroxyl group induces strong structural rearrangements, in agreement with the structural observations. Molecular electrostatic potential calculations on model systems highlight the importance of a cooperative effect in the whole hydrogen‐bond network. More precisely, the key role of the Ser142 hydroxyl group, involved in several hydrogen bonds, is underlined. Proteins 2014; 82:2303–2317. © 2014 Wiley Periodicals, Inc.     

Differential Stability of the Triple Helix of (Pro-Pro-Gly)10 in H2O and D2O: Thermodynamic and Structural Explanations

Abstract

(Pro-Pro-Gly)₁₀ [(PPG₁₀)], a collagen-like polypeptide, forms a triple-helical, polyproline-II structure in aqueous solution at temperatures somewhat lower than physiological, with a melting temperature of 24.5°C. In this article, we present circular dichroism spectra that demonstrate an increase of the melting temperature with the addition of increasing amounts of D₂O to an H₂O solution of (PPG)₁₀, with the melting temperature reaching 40°C in pure D₂O. A thermodynamic analysis of the data demonstrates that this result is due to an increasing enthalphy of unfolding in D₂O vs. H₂O. To provide a theoretical explanation for this result, we have used a model for hydration of (PPG)₁₀ that we developed previously, in which inter-chain water bridges are formed between sterically crowded waters and peptide bond carbonyls. Energy minimizations were performed upon this model using hydrogen bond parameters for water, and altered hydrogen bond parameters that reproduced the differences in carbonyl oxygen-water oxygen distances found in small-molecule crystal structures containing oxygen-oxygen hydrogen bonds between organic molecules and H₂O or D₂O. It was found that using hydrogen bond parameters that reproduced the distance typical of hydrogen bonds to D₂O resulted in a significant lowering of the potential energy of hydrated (PPG)₁₀. This lowering of the energy involved energetic terms that were only indirectly related to the altered hydrogen bond parameters, and were therefore not artifactual; the intra-(PPG₁₀) energy, plus the water-(PPG₁₀) van der Waals energy (not including hydrogen bond interactions), were lowered enough to qualitatively account for the lower enthalpy of the triple-helical conformation, relative to the unfolded state, in D₂O vs. H₂O. This result indicates that the geometry of the carbonyl-D₂O hydrogen bonds allows formation of good hydrogen bonds without making as much of an energetic sacrifice from other factors as in the case of hydration by H₂O.     

Stability and fluctuations of amide hydrogen bonds in a bacterial cytochrome c: a molecular dynamics study

Molecular dynamics (MD) simulations on a bacterial cytochrome c were performed to investigate the lifetime and fluctuations of backbone hydrogen bonds and to correlate these data with protection factors for hydrogen exchange measured by NMR spectroscopy (Bartalesi et al. in Biochemistry, 42:10923–10930, 2003). The MD simulations provide a consistent pattern in that long lifetimes of hydrogen bonds go along with small amplitude fluctuations. In agreement with experiments, differences in stability were found with a rather flexible N-terminal segment as compared with a more rigid C-terminal part. Protection factors of backbone hydrogen exchange correlate strongly with the number of contacts but also with hydrogen-bond occupancy, hydrogen-bond survival times, as well as the inverse of fluctuations of backbone atoms and hydrogen-bond lengths derived from MD simulation data. We observed a conformational transition in the C-terminal loop, and significant motion in the N-terminal loop, which can be interpreted as being the structural units involved in the onset of the protein unfolding process in agreement with experimental evidence on mitochondrial cytochrome c. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Gernot Kieseritzky and Giulia Morra both contributed equally to this work.     

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.     

Effects of backbone modification on helical peptides: The reduced carbonyl modification

Reducing a CO to a CH₂ moiety in a peptide bond destroys the ability of the peptide link to act as a proton acceptor in a hydrogen-bonded structure. Here, this modification is introduced into different positions of the helical peptide, acetyl-WGG(RAAAA)₄R-amide, and the melting of these peptides is followed using CD. Effects of this modification on helical peptides are compared to our previous N-methylation studies [C. F. Chang and M.H. Zehfus (1996) Biopolymers, Vol. 40, pp. 609–616]. While the experiments were designed to remove the same hydrogen bond from the peptide, no consistent results are obtained between these two modifications. This result suggests that these modifications not only break the backbone hydrogen bonds, but also involve other destabilizing effects. When our data is analyzed using different helix-coil transition models, the results show that as the models increase in complexity the energy associated with a single residue modification increases. Unfortunately, the most detailed dichroic model, which should best describe this system, works for only one peptide. Apparently, the models need to be further improved to better mimic our system. © 1998 John Wiley & Sons, Inc. Biopoly 46: 181–193, 1998     

The role of hydroxyproline in collagen folding: conformational energy calculations on oligopeptides containing proline and hydroxyproline

We have observed that the rate of folding of the enzymatically hydroxylated form of poly(Gly-Pro-Pro) into the triple-helical conformation is considerably higher than that of the unhydroxylated polypeptide [R. K. Chopra and V. S. Ananthanarayanan (1982) Proc. Natl. Acad. Sci. USA 79 , 7180–7184]. In this study, we examine a plausible kinetic pathway for triple-helix formation by selecting peptide models for the unhydroxylated collagen molecule, and computing their conformational energies before and after proline hydroxylation. Starting with the available data on the preferred conformations of proline- and hydroxyproline-containing peptide sequences, energy minimization was carried out on the following pairs of peptides: Gly-Ala-Pro-Gly-Ala and Gly-Ala-Hyp-Gly-Ala; Gly-Pro-Pro-Gly-Ala and Gly-Pro-Hyp-Gly-Ala; Gly-Ala-Pro-Gly-Ala-Pro and Gly-Ala-Hyp-Gly-Ala-Hyp. It was found that, with each pair of peptides, the energetically most favorable conformation (I) has an extended structure at the Gly-Ala or Gly-Pro segment and a β-bend at the Pro-Gly or Hyp-Gly segment. In the Hyp-containing peptides, this conformation is further stabilized by a (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond. Conformation I is lower in energy by about 6–13 kcal/mol of the peptide than the fully extended conformations that resemble the single collagen polypeptide chain and contain no intramolecular hydrogen bond. In contrast to the proline counterpart, the hydroxyproline-containing peptides are found capable of adopting a partially extended conformation that does not contain the β-bend but retains the (Hyp)OH…OC(Gly) hydrogen bond. The energy of this conformation is intermediate between conformation I and the fully extended conformation. The continuation of the β-bend along the chain is restricted by stereochemical constraints that are more severe in the latter two pairs of peptides than in the first pair. Such a restriction may be considered to trigger the “unbending” of the minimum energy conformation leading to its straightening into the fully extended conformation; the latter, in turn, would lead to triple-helix formation through favorable interchain interactions. We propose that the partially extended conformation in the Hyp-containing peptides could serve as a kinetic intermediate on the way to forming the fully extended conformation. Because of the (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond, this conformation would also serve to lock the trans geometry at the Gly-Ala(Pro) and Ala(Pro)-Hyp peptide bonds, thereby enhancing the rate of their helix formation. A scheme for collagen folding in proposed on the basis of these results.