Influence of local interactions on protein structure. I. Conformational energy studies of N-acetyl-N′-methylamides of pro-X and X-pro dipeptides

Conformational energy calculations using an Empirical Conformational Energy Program for Peptides (ECEPP) were carried out on the N-acetyl-N′-methylamides of Pro-X, where X = Ala, Asn, Asp, Gly, Leu, Phe, Ser, and Val, and of X-Pro, where X = Ala, Asn, Gly, and Pro. The conformational energy was minimized from starting conformations which included all combinations of low-energy single-residue minima and several standard bend structures. It was found that almost all resulting minima are combinations of low-energy single-residue minima, suggesting that intra residue interactions predominate in determining conformation. The calculations also indicate, however, that inter residue interactions can be important. In addition, librational entropy was found to influence the relative stabilities of some minima. Because of the existence of 10–100 low-energy minima for each dipeptide, the normalized statistical weight of an individual minimum rarely exceeds 0.3, suggesting that these dipeptides have considerable conformational flexibility and exist as statistical ensembles of low-energy structures. The propensity of each dipeptide to form bend conformations was calculated, and the results were compared with available experimental data. It was found that bends are favored in Pro-X dipeptides because ?_Pro is fixed by the pyrrolidine ring in a conformation which is frequently found in bends, but that bends are not favored in X-Pro dipeptides because interactions between the X residue and the pyrrolidine ring restrict the X residue to conformations which are not usually found in bends.     

Exploiting the Peptide — MHC Water Interface in the Computer-Aided Design of Non-Natural Peptides that Bind to the Class I MHC Molecule HLA-A2

Abstract

Class I major histocompatibility complex (MHC) molecules bind peptides derived from intra-cellular proteins and present them to cytotoxic T cells. Certain human immunological diseases are associated with errors in this process. Here we describe an approach to the design of non-natural peptides that could potentially interfere with peptide presentation associated with autoimmune diseases. We have shown previously that the interaction of the peptide GILGFVFTL with the MHC molecule HLA-A2 is mediated by a network of water molecules. In principle, the addition of hydroxyl groups to the peptide could allow for an enhanced interaction of the modified peptide with this water network. Here we illustrate this approach using a peptide having the non-natural amino acid homoserine at position 3, GIhSGFVFTL, and also peptides in which the Cα(F5)—CO—NH1—Cα(V6) peptide bond is replaced by an ether. Cα(F5)—CH(X)—O—Cα(V6), to give the non-natural peptide GILGF—CH(X)—O—VFTL, where X = CH₂OH or CH₃. In a 200 ps solvated molecular dynamics simulation of the HLA-A2 complexes of each peptide for GIhSGFVFTL and GILGF—CH(CH₂OH)—O—VFTL the peptide conformation remained essentially unchanged from that of GILGFVFTL in the X-ray structure of its complex with HLA-A2. In contrast, for GILGF—CH(CH₃)—O—VFTL the peptide conformation deviated from the X-ray conformation, indicating the importance of the hydroxyl group.     

Conformational energy calculations on glycosylated turns in glycoproteins

Analysis of the amino acid sequence of glycoproteins has suggested the β-turn as a likely site of glycosylation in glycoproteins. According to this model, the peptide chain traverses the interior of a globular protein, reversing its direction at the protein surface, a likely point for the attachment of hydrophilic carbohydrate residues. In order to search for plausible conformations of glycosylated β-turns in asparagine-linked glycoproteins, we have adapted the conformational energy calculation method of Scheraga and coworkers for use in carbohydrates. The parameters for nonbonded and hydrogen-bonded interactions have been published, and electrostatic parameters are derived from a CNDO calculation on a model glycopeptide. Our results indicate that the orientation of the glycosyl amide bond having the amide proton nearly trans to the anomeric proton of the sugar has the lowest energy. Although CD and nmr experiments in our laboratory have consistently found this conformation, our calculations show the conformation having these two protons in a cis relationship to lie very close in energy. Calculations on the glycopeptide linkage model, α-N-acetyl, δ-N(2-acetamido-1,2-dideoxy-β-D -glucopyranosyl)-N′-methyl-L -asparaginyl amide show that several distinct geometries are allowed for glycosylated β-turns. For a type I β-turn, three conformations of the glycosylated side chain are found within 4 kcal of the minimum, while two conformations of the glycosylated side chain are allowed for a type II turn. The hydrogen-bonded C7 conformation is also allowed. Stereoviews of the low-energy conformations reveal no major hydrogen-bonding interaction between the peptide and sugar.     

Structure and Conformation of the Antiviral Agent 5-Methoxymethyl-2′-deoxyuridine

Abstract

The three dimensional structure of the activiral agent, 5-methoxymethyl-2′-deoxyuridine (MmdUrd) was determined by x-ray diffraction methods. MMdUrd crystallized in space group P2₁2₁2₁ of the orthorhombic system with a = 9.166(1)A, b, = 25.348(1)Amm c = 5.270(1)A and Z = 4. The conformation of the glycosyl bond is anti (χ = 233.30), the deoxyribose ring has the C(2′)-endo envelope conformation (²E), the CH₂OH side chain has the g⁺ conformation and the methoxy group at the C(5) position is on the same side of pyrimidine plane as the 0(4′) oxygen. NMR spectroscopy was used to determine the conformation in solution. The spectra indicate that the sugar ring exists in a 60:40 equilibrium of the S- and N-states. The population of the three rotamers about the exocyclic c(4′)–C(5′) bond were estimated to be g⁺:t:g::61%:31%:8%. The correlaiton of molecular conforation with antiviral activity is discussed.     

Influence of local interactions on protein structure. III. Conformational energy studies of N-acety-N′-methylamides of Gly-X and X-Gly dipeptides

Conformational energy calculations using an empirical conformational energy program for peptides (ECEPP) were carried out on 20 N-acetyl- N′-methylamides of Gly-X and X-Gly depeptides, where X = Ala, Asn, Asp, Gly, Phe, Ser, Thr, Tyr, Val, and Pro, and also of Leu-Gly. Each depeptde was found to have 25 or more low-energy minima, except Gly-Thr, which had only 11 low-energy minima because of the stable side chian-backbone hydrogen present in all low-energy conformation. As a group, the stble chain-backbone hydrogen bonds present in all low-energy conformations. As a group, the Gly-containing dipeptides were calculated in all low-energy prpensity for formation of bends than the Ala-containing depeptides. The X- Gly dipeptides were calculated to favor bends more than the Gly-X dipeptides, primarlly because of the high stability of the type II bend in X-Gly dipeptides. These results are in agreement with obseved occurrences of bends in the x-ray structures of globular proteins. The calculated conformation properties were found to be in good agreement with experimental results.     

Characterization of the transition state of lysozyme unfolding. II. Effects of the intrachain crosslinking and the inhibitor binding on the transition state

The reversible refolding of a lysozyme derivative containing an extra crosslink between Glu 35 and Trp 108 was observed at pH 3.7 in solutions of 4.5M LiBr and 4.SM 1-PrOH. The rate constants of unfolding and folding for crosslinked lysozyme were compared with those for intact lysozyme. In LiBr solutions, the crosslinking greatly increases k_f but only slightly decreases k_uf. This means that the crosslinking restricts possible conformations of the U state to some conformations on the folding pathway, whereas the possible conformations of the transition state are little restricted. Although the crosslinking produces an apparently different effect on the rate constants in a PrOH solution, this could be explained by assuming that the crosslinking counteracts the effect of PrOH on the transition state. These observations suggest that the Glu 35 and Trp 108 of intact lysozyme already come into contact with each other in the course of refolding to the transition state. Kinetics of the unfolding and folding reactions of intact lysozyme were measured in the presence of 8mAf (NAG)₃. The apparent folding rate (k) was nearly equal to k_f, while the apparent unfolding rate (k) decreased sixfold. This means that the inhibitor binding stabilizes the native conformation but makes no contribution to the stabilization of the transition state. Specific interactions between (NAG)₃ and the cleft of lysozyme become available only at the last stage of folding.     

A flexible model for the cell wall polysaccharide of Streptococcus mitis J22 determined by three-dimensional 13C edited nuclear overhauser effect spectroscopy and 13C-1H long-range coupling constants combined with molecular modeling

We report on the conformation of a tetrasaccharide fragment in the repeating subunit of the cell wall polysaccharide of Streptococcus mitis J22, a receptor for the lectin of Actinomyces viscosus T14V in a bacterial coaggregation that is important in the ecological interactions of oral bacteria. Although there is considerable overlap of the ¹H-nmr signals, some cross peaks can be extracted from conventional two-dimensional nuclear Overhauser effect spectroscopy (NOESY) data on the polysaccharide. These data cannot be fit to a single conformation of the tetrasaccharide fragment. Therefore we have prepared a polysaccharide sample fully enriched in ¹³C from which we have determined accurate NOESY cross-peak volumes in a three-dimensional heteronuclear-resolved spectrum that allows accurate determination of many more NOESY cross peaks than does conventional two-dimensional spectroscopy. We have also used the ¹³C enriched polysaccharide to measure accurate values of long-range ¹³C-¹H coupling constants that can be correlated with glycosidic dihedral angles. Molecular modeling calculations on the polysaccharide fragment, including molecular dynamics simulations, identify multiple low-energy conformations. This result is to be contrasted with previous calculations on blood group oligosaccharides in our laboratory using similar methods that showed relatively rigid conformations with little flexibility of the glycosidic linkages. The present NOESY and ³J_CH data can be reconciled with a model for the antigenic tetrasaccharide in which three distinct conformations are in fast exchange. We propose that some carbohydrate epitopes such as those of the blood group oligosaccharides are relatively rigid while others such as the tetrasaccharide fragment in these studies exhibit much greater flexibility. © 1996 John Wiley & Sons, Inc.     

Conformations of model compounds of proteins. I. Acetylglycine N-methylamide

Infrared spectra in the region 4000–60 cm^?1 have been measured for acetylglycine N-methylamide and its deuterium homologs, CD₃CONHCH₂CONHCH₃, CH₃-CONHCD₂CONHCH₃, CH₂CONHCH₂CONHCD₃, and CH₃CONDCH₂CONDCH₃. Normal frequencies have also been calculated for these molecules with various conformations. The spectra show that this compound has two crystalline modifications, form A and form B. The frequencies and their isotope shifts show that the molecular conformation (Ψ, ?) of form B is near (0, 120) and that of form A near (180, 120). The short-range factors determining the conformation of peptide backbone having glycine residues are discussed.     

Crystal and molecular structure of 1′,2:4,6-di-O-isopropyl-idenesucrose tetra-acetate: a unique example of a d-fructofuranosyl ring in a sucrose derivative puckered at oxygen

Crystals of the title compound are monoclinic, space group P2₁, with cell dimensions: a = 11.260(5), b = 8.841(7), c = 15.605(6) Å, β = 102.25(7)°, and Z = 2; 2888 independent reflections, measured on a diffractometer, have been refined to R = 0.055 in the molecule, the pyranosyl ring has the expected ⁴C₁ conformation. However, the conformation of the d-fructofuranosyl ring is unexpected [P = 277.1°] with O-2′ exo to C-6′ furthest from the ring plane. The reason for this conformation, previously unknown in sucrose-related molecules, is not readily apparent from the crystal structure the eight-membered ring, however, has the expected boat-chair conformation.     

Crystal structure and solution conformation of 1-N-acetyl-β-D-glucopyranosyl amine: A model for the glycopeptide linkage

The crystal structure of the title compound, a model for the glycosyl linkage between the asparagine side chain and N-acetyl glucosamine in glycoproteins, has been determined and compared to other model structures. The pyranose ring in the crystal is in the ⁴C₁ chair conformation and the amide functions at C₁ and at C₂ are both oriented such that the amide protons are nearly trans to their respective sugar-ring protons. Coupling constants determined from the fully assigned proton nmr spectrum in aqueous solution are consistent with the conformation in the crystal.     

Nuclear magnetic resonance study of ring conformations of cyclic tetradepsipeptide AM-toxins and related peptides

Cyclic tetradepsipeptides, AM-toxin I and II, are the host-specific phytotoxins of Alternaria mali. In order to elucidate conformation-toxicity relationships, we analyzed the 270-MHz proton nmr spectra of AM-toxins and hydrogenated analogs, (D -Ala²)AM-toxin I (toxic) and (L -Ala²)AM-toxin I (not toxic), in (C²H₃)₂SO. These cyclic tetradepsipeptides do not contain N-substituted amino acid residues, and all the peptide and ester groups have been found to be transoid. Two conformers with very unequal populations have been found for AM-toxin I and II; the C^β?C^α? C?O conformations of the Dha² residues are nonplanar S-trans in the major conformer and nonplanar S-cis in the minor conformer. Only one ring conformation has been found for each of (L -Ala²) and (D -Ala²)AM-toxin I. (L -Ala²)AM-toxin I takes a C₄-type ring conformation; all the C?O groups and C^α-H bonds are oriented to the same side of the ring. (D -Ala²)AM-toxin I takes a new ring conformation; the side chain and C?O group of the L -Amp¹ residue are oriented to the same side of the ring. This new conformation is also found for the major conformers of AM-toxin I and II and thus appears to be required for the toxicity. The ring conformations of Tyr(OCH₃)¹-bearing analog tetradepsipeptides have been found to be much the same as those of Amp¹-bearing depsipeptides. Furthermore, on the basis of the two distinct conformations of (D -Ala²) and (L -Ala²)AM-toxin I, an empirical rule is proposed for the stable ring conformations of cyclic tetra-D ,L -peptides, not containing N-substituted amino acid residues.     

Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac⁴C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac⁴C prefers “proximal” orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2′, and O4′···HC(6). The “proximal” conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA^Met. The solvent accessible surface area (SASA) calculations revealed the role of ac⁴C in anticodon loop. The explicit molecular dynamics simulation study also shows the “proximal” orientation of N(4)-acetyl group. The predicted “proximal” conformation would allow ac⁴C to interact with third base of codon AUG/AUA whereas the ‘distal’ orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon–codon bases revealed that the models ac⁴C₍₃₄₎(Proximal):G₃, and ac⁴C₍₃₄₎(Proximal):A₃ are energetically more stable as compared to models ac⁴C₍₃₄₎(Distal):G₃, and ac⁴C₍₃₄₎(Distal):A₃, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor–acceptor atoms of ac⁴C₍₃₄₎(Proximal):G₃/A₃ base pairs suggesting role of ac⁴C in recognition of third letter of codons AUG/AUA. The “distal” conformation of ac⁴C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac⁴C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.     

The conformation of tetrafluorinated methyl galactoside anomers: crystallographic and NMR studies

The first single-crystal X-ray diffraction study of tetrafluorinated monosaccharide derivatives is presented. Both α- and β-methyl 2,3-dideoxy-2,2,3,3-tetrafluoro-d-galactopyranoside anomers adopt the ⁴C₁ conformation. The values for the C1–O1 and C1–O5 bond lengths and the O5–C1–O1–CH₃ dihedral angles are in line with what can be expected from the anomeric and exo-anomeric effects. The chair conformations are slightly distorted, presumably due to repulsion between 1,3-diaxial C–O and C–F bonds. The asymmetric unit of both compounds contains up to three independent molecules, which differ in the conformation of the hydroxymethyl group (including in one case a ‘forbidden’ gg rotamer). The molecular packing of the β-anomer shows a clear segregation between fluorinated and hydrophilic domains, while for the α-anomer the regions of fluorine segregation are broken by interleafing of OMe groups. There is one close OH?F contact, which is likely to arise from the crystal packing. NMR studies show that the two anomers also adopt a ⁴C₁ conformation in solution (D₂O, CDCl₃).     

Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage,a phosphodiester isostere

Modified internucleotide linkage featuring the C3′‐O‐P‐CH₂‐O‐C4″ phosphonate grouping as an isosteric alternative to the phosphodiester C3′‐O‐P‐O‐CH₂‐C4″ bond was studied in order to learn more on its stereochemical arrangement, which we showed earlier to be of prime importance for the properties of the respective oligonucleotide analogues. Two approaches were pursued: First, the attempt to prepare the model dinucleoside phosphonate with ¹³C‐labeled CH₂ group present in the modified internucleotide linkage that would allow for a more detailed evaluation of the linkage conformation by NMR spectroscopy. Second, the use of ab initio calculations along with molecular dynamics (MD) simulations in order to observe the most populated conformations and specify main structural elements governing the conformational preferences. To deal with the former aim, a novel synthesis of key labeled reagent (CH₃O)₂P(O)¹³CH₂OH for dimer preparation had to be elaborated using aqueous ¹³C‐formaldehyde. The results from both approaches were compared and found consistent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 514–529, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The chiroptical properties of proteins. II. Near-ultraviolet circular dichroism of lysozyme

A study of the near-uv CD spectrum of lysozyme was carried out in the presence and absence of the inhibitor tri-N-acetylglucosamine, and theoretical chiroptical calculations based on the tetragonal crystal structure of the enzyme and the enzyme-inhibitor complex were performed. The results of these calculations indicate that the near-uv CD spectrum of lysozyme can be adequately explained in terms of negative rotatory strengths arising from the tryptophan ¹L_a (293–300 nm) and the disulfide n-σ* bands (250 rm), and positive rotatory strength contributions from the tryptophan ¹L_b bands (291 nm) and the tyrosine ¹L_b bands (275 nm). Contributions to the rotatory strength of each band were approximated in terms of specific interactions between chromophores. It was found that the rotatory strength of most of the near-uv transitions arises primarily from coupling interactions involving other side-chain chromophores and amide groups which are in close proximity. Changes which are observed in the lysozyme CD spectrum on binding of tri-N-acetylglucosamine may be explained in terms of changes in the rotatory strength which result from interactions of the ¹L_a transitions of the active-site tryptophans with the acetamide groups of the inhibitor. The reasonable agreement which is found between the experimental and calculated rotatory strengths implies that the crystal conformation of lysozyme must resemble the solution conformation.     

A solvent effect on the side-chain conformation of phenylalanine derivatives and phenylalanine residuces in dipeptides

Three phenylalanine derivatives, Ac-Phe-NHMe, H-Phe-NHMe, and Ac-Phe-OH, were selected as models of Phe residues situated at the internal, the N-terminal, and the C-terminal positions of peptide chains, respctively. The side-chain conformations of the three compounds were analyzed from the vicnal coupling constants ³J_αβR and ³J_αβS, of their ¹H- nmr spectra measured in various organic sovlent. The two β-protons were unambiguously assined by use of sterospecifically β-monodeuterated phenylalanines. The pro-S β-proton was always situated at lower field than the pro-R one when they were observed separately. The results of a solvent effect on the conformation of the tree compounds demonstrated that the rotamer populations are remarkable sensitive of the three compounds demonstrated that the rotamer populations are remarkably sensitive to solvent polarity and that the tendencies of the solvent effects are quite different from each other. Ac-Phe-OH Showed a trend similar to that of Ac-Phe-OEt reported by early workers. The rotamer populations of other derivatives (Ac-Phe-NMe₂, Ac-Phe-NH₂, Ac-Phe-OBu^t, and Ac-Phe-OBzl) and of Phe residues in some N-acetyl dipeptde esters (Ac-Phe-Gly-OMe, Ac-Phe-Val-OMe, and Ac-Gly-Phe-OMe) were also examined in several sovent, and it was found that substituents of the Phe carboxyl group—amides or esters—determine the tendency of the solvent effect. These results are interesting in the side-chain conformations of Phe residues in peptides and proteins in an environment of low polarity can be disscussed on this experimental basis. Factors responsible for the solvent effect are discussed from (1) a structural comparison of the compunds with various carboxylic substituents, (2) an expriment with cyclohexylalanine derivatives, and (3) the measurement in mixed solvents wiht similar polarity.     

Solid-state geometry and conformation of linear,diastereoisomeric oligoprolines

We have carried out a systematic analysis of the solid-state conformational preferences of a number of linear homo-oligoprolines (to the tetramer) by ir absorption and x-ray diffraction. The peptides present different chiral sequences (tacticities), various types (urethane and amide) of N-protecting groups, and free and blocked C-termini (which imply different capabilities of forming H-bonds). The following conclusions can be drawn: (i) values for the geometry of the prolyl residue and the peptide bond in the cis and in the trans conformations are proposed; (ii) in general the conformational angles φ and ψ in the linear homo-oligoprolines have values appropriate for the polyproline II structure (conformation F); (iii) the pyrrolidine ring shows various types of puckering with no apparent relation to the backbone conformation; (iv) Pro-Pro peptide bonds generally take the trans conformation, the few cases of cis conformation being formed by Pro residues of different chirality; (v) the single H-bond donor — OH, when present, is always bonded to H-acceptors, which can be either the urethane or the amide or the peptide carbonyl but never the carbonyl group of the — COOH moiety.     

Conformational studies of N-acetyl-N′-methylamide derivatives of α-aminobutyric acid,norvaline, and valine. I. Preferred conformations in solution as studied by 1H-nmr spectroscopy

The ¹H-nmr studies were extensively carried out to elucidate preferred conformations of dipeptides CH₃C*O—X—NHCH₃, with X = Abu, nVal, and Val in various solvents. The vicinal ¹H—¹H coupling constants for the NH—C^αH moiety and those around the C^α—C^β bond in the articulated side chain provided the information regarding the average conformation of these molecules. The results indicate that transformation of skeletal conformations takes place in solution among conformers having similar dihedral angles, θ, in the Karplus expression.     

A conformational study of the tetrapeptide CH3CO-Ala-Asp-Gly-Lys-NHCH3 corresponding to a β-bend in staphylococcal nuclease

The tetrapeptide sequence Ala-Asp-Gly-Lys occurs as a type I′ β-bend at residues 94–97 in staphylococcal nuclease. We have synthesized theN-acetyl,N′-methylamide derivative of this tetrapeptide and studied its conformation in solution, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. In the synthesis, special attention was paid to the possibility of cyclic aspartimide formation giving rise to mixtures of α- and β-Asp-Gly products. The presence of such a mixture was excluded by infrared, NMR, and other analytical procedures applied to the products and to models for α- and β-linked aspartyl residues. The CD spectra of the protected tetrapeptide in water, methanol, and trifluoroethanol show no evidence of preferred chain conformations. In dimethylsulfoxide-d ₆ , however, the NMR spectra are consistent with the presence of a population of conformers in which the Lys and C-terminal NHCH₃ amide protons are shielded from solvent. Taken together with the observed³J_NH-C ^α _H coupling constants for all residues, this permitted the construction and energetic evaluation of possible conformations in solution. Only one such conformation was fully compatible with the NMR data; this is a type II β-bend in which the Lys and C-terminal NHCH₃ amide protons are close to the Ala C=O group and may form bifurcated hydrogen bonds with it. This conformation can be converted into the conformation existing in staphylococcal nuclease by rotating the plane of the Ala-Asp peptide group by about 120° around a line connecting the Ala and Asp C_α atoms and by making small shifts in dihedral angles elsewhere in the peptide.