On the Bioactive Conformation of a Small Peptide and its Set of Thermodynamically Accessible Conformations

Abstract

In order to investigate the relationship between the bioactive conformation of a peptide and its set of thermodynamically accessible structures in solution, the conformational profile of the tetrapeptide Ac-Pro-Ala-Pro-Tyr-OH was characterized by computational methods. Search of the conformational space was performed within the molecular mechanics framework using the AMBER4.0 force field with an effective dielectric constant of 80. Unique structures of the peptide were compared with its bioactive conformation for the protein Streptomyces griseus Protease A, as taken from the crystal structure of the enzyme-peptide complex. The results show that the bound conformation is close to one of the unique conformations characterized in the conformational search of the isolated peptide. Moreover, the lowest energy minimum characterized in the conformational search exhibits large deviations when compared to the bound conformation of the crystal structure.     

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.     

Studies on hydrogen bonds. Part V--Hydrogen bonding in energy minimization studies of peptides

An energy term, representing the N-H...O type of hydrogen bond, which is a function of the hydrogen bond length (R) and angle (theta) has been introduced in an energy minimization program, taking into consideration its interpolation with the non-bonded energy for borderline values of R and theta. The details of the mathematical formulation of the derivatives of the hydrogen bond function as applicable to the energy minimization have been given. The minimization technique has been applied to hydrogen bonded two and three linked peptide units (gamma-turns and beta-turns), and having Gly, Ala and Pro side chains. Some of the conformational highlights of the resulting minimum energy conformations are a) the occurrence of the expected 4----1 hydrogen bond in all of the burn-turn tripeptide sequences and b) the presence of an additional 3----1 hydrogen bond in some of the type I and II tripeptides with the hydrogen bonding scheme in such type I beta-turns occurring in a bifurcated form. These and other conformational features have been discussed in the light of experimental evidence and theoretical predictions of other workers.     

Channel structures in synthetic polypeptides with alternating configurations. Conformational analysis of poly(DL-proline)

Theoretical conformational analysis of L,D alternating sequences of poly alpha-amino acids is reported in connection with the ability of naturally occurring peptide and depsipeptide having alternating configurations to increase selectively the ion permeability across membranes. The most stable structures of poly(DL-proline), of which the conformational variability is practically limited to the choice between cis and trans conformations of the peptide bonds, were characterized. The all-trans conformation results in a flat helical structure possessing the main features for acting as an ion channel across membranes as actually found experimentally. Random cis-trans conformational sequences provide an alternative mechanism of ion transport intermediate between the ion channel and the ion carrier.     

Solid state and solution conformations of a hybrid alphagammaalphaalphagammaalpha hexapeptide. Characterization of a backbone expanded analog of the alpha-polypeptide 3(10)-helix

The stereochemically constrained gamma amino acid residue gabapentin (1-(aminomethyl)cyclohexaneacetic acid, Gpn) has been incorporated into a host alpha-peptide sequence. The structure of a hybrid alphagammaalphaalphagammaalpha peptide, Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe in crystals reveals a continuous helical conformation stabilized by three intramolecular 4 --> 1 C(12) hydrogen bonds across the alphagamma/alphagamma segments and one C(10) hydrogen bond across the central alphaalpha segment. This conformation corresponds to an expanded analog of the canonical all-alpha polypeptide 3(10)-helix, with insertion of two additional backbone atoms at each gamma residue. Solvent dependence of NH chemical shifts in CDCl(3) solution are consistent with conformation in which the NH groups of Aib (3), Leu (4), Gpn (5), and Aib (6) are hydrogen bonded, a feature observed in the solid state. The nonsequential NOEs between Gpn (2) NH <--> Leu (4) NH and Gpn (2) NH <--> Gpn (5) NH support the presence of additional conformations in solution. Temperature-dependent line broadening of NH resonances confirms the occurrence of rapid exchange between multiple conformations at room temperature. Two conformational models which rationalize the observed nonsequential NOEs are presented, both of which contain three hydrogen bonds and are consistent with the known stereochemical preferences of the Gpn residue.     

Methods of peptide conformation studies

In solution most of the peptides assume multiple flexible conformations. Determination of the dominant conformers and evaluation of their populations is the aim of peptide conformation studies, in which theoretical and experimental methods play complementary roles. Molecular dynamics or Monte Carlo methods are quite effective in searching the conformational space accessible to a peptide but they are not able to estimate, precisely enough, the populations of various conformations. Therefore, they must be supplemented by experimental data. In this paper, a short review of the experimental methods, most widely used in peptide conformational studies, is presented. Among them NMR plays the leading role. Valuable information is also obtained from hydrogen exchange, fluorescence resonance energy transfer, and circular dichroism measurements. The advantages and shortcomings of these methods are discussed.     

Design of peptides with branched beta-carbon dehydro-residues: syntheses, crystal structures and molecular conformations of two peptides, (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3.

Highly specific structures can be designed by inserting dehydro-residues into peptide sequences. The conformational preferences of branched beta-carbon residues are known to be different from other residues. As an implication it was expected that the branched beta-carbon dehydro-residues would also induce different conformations when substituted in peptides. So far, the design of peptides with branched beta-carbon dehydro-residues at (i + 1) position has not been reported. It may be recalled that the nonbranched beta-carbon residues induced beta-turn II conformation when placed at (i + 2) position while branched beta-carbon residues induced beta-turn III conformation. However, the conformation of a peptide with a nonbranched beta-carbon residue when placed at (i + 1) position was not found to be unique as it depended on the stereochemical nature of its neighbouring residues. Therefore, in order to induce predictably unique structures with dehydro-residues at (i + 1) position, we have introduced branched beta-carbon dehydro-residues instead of nonbranched beta-carbon residues and synthesized two peptides: (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3 with DeltaVal and DeltaIle, respectively. The crystal structures of peptides (I) and (II) have been determined and refined to R-factors of 0.065 and 0.063, respectively. The structures of both peptides were essentially similar. Both peptides adopted type II beta-turn conformations with torsion angles; (I): phi1 = -38.7 (4) degrees, psi1 = 126.0 (3) degrees; phi2 = 91.6 (3) degrees, psi2 = -9.5 (4) degrees and (II): phi1 = -37.0 (6) degrees, psi1 = 123.6 (4) degrees, phi2 = 93.4 (4), psi2 = -11.0(4) degrees respectively. Both peptide structures were stabilized by intramolecular 4-->1 hydrogen bonds. The molecular packing in both crystal structures were stabilized in each by two identical hydrogen bonds N1...O1' (-x, y + 1/2, -z) and N2...O2' (-x + 1, y + 1/2, -z) and van der Waals interactions.     

Conformational analysis of the 20-residue membrane-bound portion of melittin by conformational space annealing

The conformational space of the 20-residue membrane-bound portion of melittin has been investigated extensively with the conformational space annealing (CSA) method and the ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm. Starting from random conformations, the CSA method finds that there are at least five different classes of conformations, within 4 kcal/mol, which have distinct backbone structures. We find that the lowest energy conformation of this peptide from previous investigations is not the global minimum-energy conformation (GMEC); but it belongs to the second lowest energy class of the five classes found here. In four independent runs, one conformation is found repeatedly as the lowest energy conformation of the peptide (two of the four lowest energy conformations are identical; the other two have essentially identical backbone conformations but slightly different side-chain conformations). We propose this conformation, whose energy is lower than that found previously by 1.9 kcal/mol, as the GMEC of the ECEPP/3 force field. The structure of the proposed GMEC is less helical and more compact than the previous one. It appears that the CSA method can find several classes of conformations of a 20-residue peptide starting from random conformations utilizing only its amino acid sequence information. The proposed GMEC has also been found with a modified electrostatically driven Monte Carlo method [D. R. Ripoll, A. Liwo, and H.A. Scheraga (1998) “New Developments of the Electrostatically Driven Monte Carlo Method: Test on the Membrane-Bound Portion of Melittin,” Biopolymers, Vol. 46, pp. 117–126]. © 1998 John Wiley & Sons, Inc. Biopoly 46: 103–115, 1998     

Statistical Mechanical Approach for Predicting the Transition to Non-B DNA Structures in Supercoiled DNA

Abstract

Supercoiling causes global twist of DNA structure and the supercoiled state has wide influence on conformational transition. A statistical mechanical approach was made for prediction of the transition probability to non-B DNA structures under torsional stress. A conditional partition function was defined as the sum over all possible states of the DNA sequence with basepair 1 and basepair n being in B-form helix and a recurrence formula was developed which expressed the partition function for basepair n with those for less number of pairs. This new definition permits a quick enumeration of every configuration of secondary structures. Energetic parameters of all conformations concerned, involving B-form, interior loop, cruciform and Z-form, were included in the equation. The probability of transition to each non-B conformation could be derived from these conditional partition functions. For treatment of effects of superhelicity, supercoiling energy was considered, and a twist of each conformation was determined to minimize the supercoiling energy. As the twist itself affects the transition probability, the whole scheme of equations was solved by renormalization technique. The present method permits a simultaneous treatment of serveral types of conformations under a common torsional stress.

A set of energetic parameters of DNA secondary structures has been chosen for calculation. Some DNA sequences were submitted to the calculation, and all the sequences that we submitted gave stable convergence. Some of them have been investigated the critical supercoil density for the transition to non-B DNA structures. Even though the reliability of the set of parameters was not enough, the prediction of secondary structure transition showed good agreement with reported observation. Hence, the present algorithm can estimate the probability of local conformational change of DNA under a given supercoil density, and also be employed to predict some specific sequences in which conformational change is sensitive to superhelicity.     

Conformational studies on somatostatin. II. The C-terminal hexapeptide fragment

The results of a conformational study on the C terminal hexapeptide of Somatostatin are presented. Semi-empirical energy calculations and high resolution NMR methods have been used to obtain information on the conformational properties of SRIF9-14 in [2H6]dimethylsulfoxide and 2H2O. It is concluded from the energy calculations that the peptide has an averaged conformation in which semi extended and folded structures are important. Only some of the folded conformations can explain the chemical shift differences between the amino acid residues Thr10 and Thr12 as a ring current shift by the Phe11 aromatic ring on Thr10. The nonequivalence is more pronounced in dimethyl-sulfoxide (0.23--0.15 ppm) where it decreases with increasing temperature towards the temperature independent value in 2H2O (0.03 ppm). This suggests that the folded conformations are somewhat predominant in dimethylsulfoxide solutions. In 2H2O the semi extended and folded structures are statistically equally important and the peptide is more flexible. A comparison with a study on the smaller fragments SRIF10-12 and SRIF10-13 which have similar conformational properties, demonstrates the usefulness of the fragment approach in conformational studies of peptides.     

A new proposal for the primary and secondary structure of the glycan moiety of pseudomurein. Conformational energy calculations on the glycan strands with talosaminuronic acid in 1C conformation and comparison with murein

Using the method of conformational energy calculations, favoured conformations of a pseudomurein sugar strand built up from beta 1,3-linked N-acetyl-D-glucosamine and N-acetyl-L-talosaminuronic acid were obtained. Such a completely beta 1,3-linked polysaccharide primary structure, although contrasting with the originally proposed alternating beta 1,3-alpha 1,3-linked structure [K?nig, H., Kandler, O., Jensen, M. and Rietschel, E. Th. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 627-636] would be in agreement with all experimental data hitherto known. Starting from an analysis of favoured conformations of the monosaccharide building blocks and those obtained for disaccharide parts, the favoured helical conformations of the complete polysaccharide chains could be explored. Our completely beta 1,3-linked chain could adopt two types of conformation: extended and hollow; the latter was discarded as unsuitable for cell wall assembly. The extended conformation type was shown to exhibit a remarkable similarity if compared to the secondary structures accessible to murein-type polysaccharide chains. In contrast to the conformations accessible for the beta 1,3-alpha 1,3-linked primary structure, the new hypothetical pseudomurein structure was found to be more extended, possessed a flexible peptide attachment site at every second sugar residue and led to an orientation of consecutive peptide attachment sites analogous to the data known for murein-type chains. From the two possibilities compatible with the experimental data available, the completely beta 1,3-linked sugar strand structure could well be realized in the native pseudomurein network of methanobacteria. In this case the hypothesis for a similar three-dimensional architecture for murein and pseudomurein would be supported.     

Spatial structure of the rp142 peptide containing the immunodominant epitope of the HIV-1 gp120 protein. A theoretical study]

A model of spatial structure of the synthetic peptide rp142 (24 amino acid residues) containing the immunodominant epitope of the HIV-1 protein gp120 in the region Gly-10-Phe-15 was constructed by the method of "constrained" molecular mechanics, which uses the algorithms of theoretical conformational analysis, based on NMR spectroscopy data. A comparative analysis of calculated conformations revealed that the spatial structure of rp142 in solution can be described by a family of conformations to which nine different structural clusters involving the sets of topologically close conformers correspond. It is shown that the main chain of the peptide forms irregular but "structured" conformations in which the main portion of amino acid residues is incorporated into beta-turns and helix-like fragments, while Pro-11 and Gly-12 form in some structures inverse gamma-turns, which rarely occur in protein-peptide molecules. It was found that the spatial packing of the Gly-10-Phe-15 hexapeptide in different clusters is realized at different internal rotation angles, to which topologically close structures correspond. It is assumed that this invariant structural element describes the "conformation of complex formation" that is complementary to the antigen-binding center of antibodies and is responsible for their binding to the peptide.     

Conformations of folded proteins in restricted spaces

A new method is presented to examine the complete range of folded topologies accessible in the compact state of globular proteins. The procedure is to generate all conformations, with volume exclusion, upon a lattice in a space restricted to the individual protein's known compact conformational space. Using one lattice point per residue, we find 10(2)-10(4) possible compact conformations for the five small globular proteins studied. Subsequently, these conformations are evaluated in terms of residue-specific, pairwise contact energies that favor nonbonded, hydrophobic interactions. Native structures for the five proteins are always found within the best 2% of all conformers generated. This novel method is simple and general and can be used to determine a small group of most favorable overall arrangements for the folding of specific amino acid sequences within a restricted space.     

Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer's disease. Analysis of circular dichroism spectra.

The A4 or beta-peptide (39 to 43 amino acid residues) is the principal proteinaceous component of amyloid deposits in Alzheimer's disease. Using circular dichroism (c.d.), we have studied the secondary structures and aggregational properties in solution of 4 synthetic amyloid beta-peptides: beta-(1-28), beta-(1-39), beta-(1-42) and beta-(29-42). The natural components of cerebrovascular deposits and extracellular amyloid plaques are beta-(1-39) and beta-(1-42), while beta-(1-28) and beta-(29-42) are unnatural fragments. The beta-(1-28), beta-(1-39) and beta-(1-42) peptides adopt mixtures of beta-sheet, alpha-helix and random coil structures, with the relative proportions of each secondary structure being strongly dependent upon the solution conditions. In aqueous solution, beta-sheet structure is favored for the beta-(1-39) and beta-(1-42) peptides, while in aqueous solution containing trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), alpha-helical structure is favored for all 3 peptides. The alpha-helical structure unfolds with increasing temperature and is favored at pH 1 to 4 and pH 7 to 10; the beta-sheet conformation is temperature insensitive and is favored at pH 4 to 7. Peptide concentration studies showed that the beta-sheet conformation is oligomeric (intermolecular), whereas the alpha-helical conformation is monomeric (intramolecular). The rate of aggregation to the oligomeric beta-sheet structure (alpha-helix----random coil----beta-sheet) is also dependent upon the solution conditions such as the pH and peptide concentration; maximum beta-sheet formation occurs at pH 5.4. These results suggest that beta-peptide is not an intrinsically insoluble peptide. Thus, solution abnormalities, together with localized high peptide concentrations, which may occur in Alzheimer's disease, may contribute to the formation of amyloid plaques. The hydrophobic beta-(29-42) peptide adopts exclusively an intermolecular beta-sheet conformation in aqueous solution despite changes in temperature or pH. Therefore, this segment may be the first region of the beta-peptide to aggregate and may direct the folding of the complete beta-peptide to produce the beta-pleated sheet structure found in amyloid deposits. Differences between the solution conformations of the beta-(1-39) and beta-(1-42) peptides suggests that the last 3 C-terminal amino acids are crucial to amyloid deposition.     

Human telomere quadruplex: refolding and selection of individual conformers via RNA/DNA chimeric editing

The conformation of the guanine quadruplex formed by the human telomere (HT) repeat in solutions containing physiological concentrations of K+ ions has been a topic of intensive investigation during the past several years. Of particular interest are the directionality of the overall folding pattern, i.e., parallel, antiparallel, or a combination of these two modes, and the alternation, if any, of the glycosidic bond conformation between syn and anti. An additional issue involves resolving mixtures of conformations when more than one species is present. We approach these questions using selective substitution of riboguanosine, rG, for deoxyriboguanosine, dG. Using a combination of circular dichroism, gel electrophoresis, equilibrium ultracentrifugation, and imino proton NMR, we are able to show that these modifications can yield sequences which fold into parallel or antiparallel conformations consisting of one or two strands. We also demonstrate that chimeric editing of the HT sequence permits isolating one of two conformational isomers existing in solution in the presence of KCl. The ability to engineer and control quadruplex folding motifs illustrated here with HT may prove useful more generally for a variety of quadruplex-forming sequences.     

Assessing the native state conformational distribution of ubiquitin by peptide acidity

At equilibrium, every energetically feasible conformation of a protein occurs with a non-zero probability. Quantitative analysis of protein flexibility is thus synonymous with determining the proper Boltzmann-weighting of this conformational distribution. The exchange reactivity of solvent-exposed amide hydrogens greatly varies with conformation, while the short-lived peptide anion intermediate implies an insensitivity to the dynamics of conformational motion. Amides that are well-exposed in model conformational ensembles of ubiquitin vary a million-fold in exchange rates which continuum dielectric methods can predict with an rmsd of 3. However, the exchange rates for many of the more rarely exposed amides are markedly overestimated in the PDB-deposited 2K39 and 2KN5 ubiquitin ensembles, while the 2NR2 ensemble predictions are largely consistent with those of the Boltzmann-weighted conformational distribution sampled at the level of 1%. The correlation between the fraction of solvent-accessible conformations for a given amide hydrogen and the exchange rate constant for that residue provides a useful monitor of the degree of completeness with which a given ensemble has sampled the energetically accessible conformational space. These exchange predictions correlate with the degree to which each ensemble deviates from a set of 46 ubiquitin X-ray structures. Kolmogorov-Smirnov analysis for the distribution of intra- and inter-ensemble pairwise structural rmsd values assisted the identification of a subensemble of 2K39 that eliminates the overestimations of hydrogen exchange rates observed for the full ensemble. The relative merits of incorporating experimental restraints into the conformational sampling process are compared to using these restraints as filters to select subpopulations consistent with the experimental data.     

An approach to the problem of the structuro-functional organization of natural peptides

Theory and computational scheme of three-dimensional structure and dynamic conformational properties of naturally occurring peptides are proposed basing on a known amino acid sequence. The diverse biological activity of a low-molecular peptide is shown to arise from a restricted number of preferable spatial structures which may occur under physiological conditions. Each particular function of an oligopeptide is connected to a definite spatial structure, belonging to the set of low-energy conformations from one biological activity of a peptide shift of the conformational equilibrium caused by a change of environmental conditions. This shift is provided for by specific intramolecular interactions, alternative in their nature, which stabilize a particular structure. An approach is suggested which enables to construct a synthetic analog with the predetermined physiologically active conformation, prior to all chemical and biological tests.     

Protein local conformations arise from a mixture of Gaussian distributions

The classical approaches for protein structure prediction rely either on homology of the protein sequence with a template structure or on ab initio calculations for energy minimization. These methods suffer from disadvantages such as the lack of availability of homologous template structures or intractably large conformational search space, respectively. The recently proposed fragment library based approaches first predict the local structures,which can be used in conjunction with the classical approaches of protein structure prediction. The accuracy of the predictions is dependent on the quality of the fragment library. In this work, we have constructed a library of local conformation classes purely based on geometric similarity. The local conformations are represented using Geometric Invariants, properties that remain unchanged under transformations such as translation and rotation, followed by dimension reduction via principal component analysis. The local conformations are then modeled as a mixture of Gaussian probability distribution functions (PDF). Each one of the Gaussian PDF's corresponds to a conformational class with the centroid representing the average structure of that class. We find 46 classes when we use an octapeptide as a unit of local conformation. The protein 3-D structure can now be described as a sequence of local conformational classes. Further, it was of interest to see whether the local conformations can be predicted from the amino acid sequences. To that end,we have analyzed the correlation between sequence features and the conformational classes.     

Reconstruction of protein backbones from the BriX collection of canonical protein fragments

As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more than 1,000 frequently observed conformations per peptide length for 6 different variation levels. Analysis of the performance of BriX revealed an average structural coverage of protein structures of more than 99% within a root mean square distance (RMSD) of 1 Angstrom. Globally, we are able to reconstruct protein structures with an average accuracy of 0.48 Angstrom RMSD. As expected, regular structures are well covered, but, interestingly, many loop regions that appear irregular at first glance are also found to form a recurrent structural motif, albeit with lower frequency of occurrence than regular secondary structures. Larger loop regions could be completely reconstructed from smaller recurrent elements, between 4 and 8 residues long. Finally, we observed that a significant amount of short sequences tend to display strong structural ambiguity between alpha helix and extended conformations. When the sequence length increases, this so-called sequence plasticity is no longer observed, illustrating the context dependency of polypeptide structures.     

三肽和四肽构象空间的可视化方法

研究蛋白质寡肽构象在构象空间中的分布情况,对提取寡肽模式并构建短肽库具有重要意义。通过构建一个保距映射,将以主链原子均方根距离(root mean square distance,RMSD)为距离测度的三肽构象空间变换为一维直线上的欧氏距离空间,从而直观地展现三肽构象的聚集情况,表明三肽主链构象可以用单一变量编码。应用该特性对四肽的构象空间加以分析,将四肽构象映射到三维空间中,从而以可视的方式描述四肽构象空间的聚集情况。对短肽构象空间的初步分析表明,短肽的聚集性和二级结构有着密切的联系。在四肽构象空间中存在有自然边界的离散区域(与螺旋等结构相关),也有一些区域(与折叠等结构有关)难以进一步划分。这种方法也为以可视方式分析高维空间中肽段的聚集性给出了一种可能的方案。