Prediction of the native conformation of a polypeptide by a statistical-mechanical procedure. I. Backbone structure of enkephalin

A new methodology for theoretically predicting the native, three-dimensional structure of a polypeptide is presented. Based on equilibrium statistical mechanics, an algorithm has been designed to determine the probable conformation of a polypeptide by calculating conditional free-energy maps for each residue of the macromolecule. The conditional free-energy map of each residue is computed from a set of probability integrals, obtained by summing over the interaction energies of all pairs of nonbonded atoms of the whole molecule. By locating the region(s) of lowest free energy for each map, the probable conformation for each residue can be identified. The native structure of the polypeptide is assumed to be the combination of the probable conformations of the individual residues. All multidimensional probability integrals are evaluated by an adaptive Monte Carlo algorithm (SMAPPS —Statistical-Mechanical Algorithm for Predicting Protein Structure). The Monte Carlo algorithm searches the entire conformational space, adjusting itself automatically to concentrate its sampling in regions where the magnitude of the integrand is largest (“importance sampling”). No assumptions are made about the native conformation. The only prior knowledge necessary for the prediction of the native conformation is the amino acid sequence of the polypeptide. To test the effectiveness of the algorithm, SMAPPS was applied to the prediction of the native conformation of the backbone of Met-enkephalin, a pentapeptide. In the calculations, only the backbone dihedral angles (? and ψ) were allowed to vary; all side-chain (χ) and peptide-bond (ω) dihedral angles were kept fixed at the values corresponding to the alleged global minimum energy previously determined by direct energy minimization. For each conformation generated randomly by the Monte Carlo algorithm, the total conformational energy of the polypeptide was obtained from established empirical potential energy functions. Solvent effects were not included in the computations. With this initial application of SMAPPS , three distinct low-free-energy β-bend structures of Met-enkephalin were found. In particular, one of the structures has a conformation remarkably similar to the one associated with the previously alleged global minimum energy. The two additional structures of the pentapeptide have conformational energies lower than the previously computed low-energy structure. However, the Monte Carlo results are in agreement with an improved energy-minimization procedure. These initial results on the backbone structure of Met-enkephalin indicate that an equilibrium statistical-mechanical procedure, coupled with an adaptive Monte Carlo algorithm, can overcome many of the problems associated with the standard methods of direct energy minimization.     

Prediction of the native conformation of a polypeptide by a statistical-mechanical procedure. II. Average backbone structure of enkephalin

The average conformation of Met-enkephalin was determined by using an adaptive, importance-sampling Monte Carlo algorithm (SMAPPS—Statistical Mechanical Algorithm for Predicting Protein Structure). In the calculation, only the backbone dihedral angles (? and ψ) were allowed to vary; i.e., all side-chain (χ) and peptide-bond (ω) dihedral angles were kept fixed at the values corresponding to a low-energy structure of the pentapeptide. The total conformational energy for each randomly generated structure of the polypeptide was obtained by summing over the interaction energies of all pairs of nonbonded atoms of the whole molecule. The interaction energies were computed by the program ECEPP/2 (Empirical Conformational Energy Program for Peptides). Solvent effects were not included in the computation. The calculation was repeated until a total of 10 independent average conformations were established. The regions of conformational space occupied by the average structures were compared with the regions of low conditional free energy obtained by SMAPPS in the first paper of this series. Such a comparison provides an analysis of the capacity of SMAPPS to adjust the Monte Carlo search to regions of highest probability. The results demonstrate that the ability of SMAPPS to focus the Monte Carlo search is excellent. Finally, the 10 independent average conformations and the mean of the 10 average structures were utilized as the initial conformations for a direct energy minimization of the pentapeptide. Of the 11 final energy-minimized structures, three of the conformations were found to be equivalent to the conformation of lowest energy determined previously. In addition, all but two of the remaining energy-minimized structures were found to correspond to one of the two other conformations of high probability obtained in the first paper of this series. These results indicate that a set of independent average conformations can provide a rational, unbiased choice for the initial conformation, to be used in a direct energy minimization of a polypeptide. The final energy-minimized structures consequently constitute a set of low-energy conformations, which include the global energy minimum.     

Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors. III. Probable binding conformations of mu-agonists with phenylalanine in position 3.

Theoretical conformational analysis was carried out for several tetrapeptide analogues of beta-casomorphin and dermorphin containing a Phe residue in position 3. Sets of low-energy backbone structures of the mu-selective peptides [N-Me-Phe3, D-Pro4]-morphiceptin and Tyr-D-Orn-Phe-Asp-NH2 were obtained. These sets of structures were compared for geometrical similarity between themselves and with the low-energy conformations found for the delta-selective peptide Tyr-D-Cys-Phe-D-Pen-OH and nonactive peptide Tyr-Orn-Phe-Asp-NH2. Two pairs of geometrically similar conformations of mu-selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the mu-receptor, were selected as two alternative models of probable mu-receptor-bound backbone conformations. Both models share geometrical similarity with the low-energy structures of the linear mu-selective peptide Tyr-D-Ala-Phe-Phe-NH2. Putative binding conformations of Tyr1 and Phe3 side chains are also discussed.     

Methionine enkephalin and isosteric analogues. Part II.: Receptor conformation of methionine enkephalin

Biological activities are reported for two different types of analogues of methionine enkephalin. Cyclic analogues, bridged between the amino- and carboxy- terminals of the parent peptide, are inactive. In contrast, significant levels of activity are displayed by linear isosterically modified analogues in which the Tyr1-Gly2 peptide bond is replaced by either -CH2NH- or -CH2CH2-. Similar replacements of the Gly2-Gly3 peptide bond yield compounds with much reduced potency. These modifications serve as useful probes of the receptor conformation. Based on these findings, a model is proposed for interaction between enkephalin and its receptor.     

Structural organization of [met]enkephalin and endorphin molecules. III. Theoretical conformation analysis of beta-endorphin

Conformational energy calculations were carried out for beta-endorphin. Its spatial structure can be described by nine low-energy conformations. The calculations yielded the values of all dihedral angles of the backbone and side chains of these forms as well as intra- and inter-residue interaction energies.     

Regeneration of native ovalbumin conformation disrupted by citraconylation.

Ovalbumin was reacted with a 960-fold molar excess of citraconic anhydride, and 91% of the epsilon-amino groups, representing 18 of the 20 lysine residues, were citraconylated. As detected by fluorescence and far-ultraviolet circular dichroic (CD) measurements, the modified protein displayed significant disruption of the native conformation. Treatment at pH 2.2 for 5 h resulted in the hydrolysis of 10 of the 18 citraconyl groups, but when subjected to the acid conditions for 12 h, all 18 modifying groups were removed. Electrophoretically, the 5-h and the 12-h acid-treated proteins were homogeneous and showed decreasing anodic mobility at pH 8.3; indeed, the anodic mobility of the 12-h acid-treated protein was identical to that of the native protein. Similarly, the 12-h acid-treated protein possessed conformational properties almost indistinguishable from the native protein. These properties included similar emission fluorescence spectra and far-ultraviolet CD spectra, similar resistance to undergoing helix-to-coil transition at pH 12.2, and identical urea unfolding curves, and thus identical urea transition mid-point of about 8.0 M. These observations indicate that the protein with all the epsilon-amino groups regenerated by acid treatment has the same conformational stability as the native ovalbumin.     

Prediction of protein conformation on the basis of a search for compact structures: test on avian pancreatic polypeptide.

Based on the concept that hydrophobic interactions cause a polypeptide chain to adopt a compact structure, a method is proposed to predict the structure of a protein. The procedure is carried out in four stages: (1) use of a virtual-bond united-residue approximation with the side chains represented by spheres to search conformational space extensively using specially designed interactions to lead to a collapsed structure, (2) conversion of the lowest-energy virtual-bond united-residue chain to one with a real polypeptide backbone, with optimization of the hydrogen-bond network among the backbone groups, (3) perturbation of the latter structure by the electrostatically driven Monte Carlo (EDMC) procedure, and (4) conversion of the spherical representation of the side chains to real groups and perturbation of the whole molecule by the EDMC procedure using the empirical conformational energy program for peptides (ECEPP/2) energy function plus hydration. Application of this procedure to the 36-residue avian pancreatic polypeptide led to a structure that resembled the one determined by X-ray crystallography; it had an alpha-helix starting at residue 13, with the N-terminal portion of the chain in an extended conformation packed against the alpha-helix. Similar structures with slightly higher energies, but looser packing, were also obtained.     

Multiple conformations of the sea anemone polypeptide anthopleurin-A in solution.

Anthopleurin-A (AP-A) is a member of a family of sea anemone-derived polypeptides that interact with sodium channels in a voltage-dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3-dimensional structure of AP-A were complicated by the fact that it was found to exist in 2 conformations in solution. Using 1H- and 13C-NMR spectroscopy, we have now shown that this conformational heterogeneity arises from cis-trans isomerization about the Gly 40-Pro 41 peptide bond and that in the major form of the protein this peptide bond adopts a cis conformation. Furthermore, the increased sensitivity afforded by higher-field NMR has allowed identification of additional minor conformations of AP-A, the origin of which is presently unknown. We believe there will be many more examples of the detection by high-field NMR of previously unobserved minor conformations of proteins in solution.     

Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation

Current drug discovery efforts focus primarily on proteins with defined enzymatic or small molecule binding sites. Autoregulatory domains represent attractive alternative targets for small molecule inhibitors because they also occur in noncatalytic proteins and because allosteric inhibitors may avoid specificity problems inherent in active site-directed inhibitors. We report here the identification of wiskostatin, a chemical inhibitor of the neural Wiskott-Aldrich syndrome protein (N-WASP). Wiskostatin interacts with a cleft in the regulatory GTPase-binding domain (GBD) of WASP in the solution structure of the complex. Wiskostatin induces folding of the isolated, unstructured GBD into its autoinhibited conformation, suggesting that wiskostatin functions by stabilizing N-WASP in its autoinhibited state. The use of small molecules to bias conformational equilibria represents a potentially general strategy for chemical inhibition of autoinhibited proteins, even in cases where such sites have not been naturally evolved in a target.     

Avian pancreatic polypeptide fragments refold to native aPP conformation when combined in solution: a CD and VCD study

An equimolar mixture of avian pancreatic polypeptide (aPP) fragments aPP(1-11)-NH2 and Ac-aPP(12-36) had an electronic circular dichroism (ECD) spectrum that was similar to that of whole aPP in H2O and even more so in 30% (v/v) trifluoroethanol (TFE) in 15 mM Na2HPO4, but was different from the sum of the spectra of the individual fragments. The vibrational circular dichroism (VCD) spectrum of the combined fragments in 30% (v/v) TFE in 15 mM Na2HPO4 in D2O was also similar to that of the intact aPP and unlike the sum of the VCD spectra of the fragments. The interaction of these fragments is thus sufficient to support the conformation of whole aPP. This study demonstrates that VCD, in combination with ECD, is useful for the study of protein-protein interactions.     

Prediction of the conformation of the histones.

The secondary structures of the histones, H1, H2A, H2B, H3, and H4 have been predicted utilizing the predictive scheme of Chou and Fasman (Biochemistry 13:211, 222[1974]) and a new set of conformational parameters based on the X-ray data of 29 protein structures. The alpha-helical, beta-sheet, reverse beta-turns, and random coil regions of these proteins are carefully delineated. Structures are specified which are most probably under various environmental conditions, i.e., for changes in ionic strength, association between histones and in association with DNA. Potential conformational changes within these histones are also predicted.     

Structural organization of [Met]enkephalin and endorphin molecules. I. Theoretical conformation analysis of [Met]enkephalin

Using a semi-empirical method, an a priori conformational analysis of the [Met]-enkephalin molecule was carried out. Calculations yielded the values of all dihedral angles of the backbone and side chains of the peptide's forms as well as intra- and inter-residue interaction energies.     

Water and polypeptide conformations in the gramicidin channel. A molecular dynamics study.

Theoretical studies of ion channels address several important questions. The mechanism of ion transport, the role of water structure, the fluctuations of the protein channel itself, and the influence of structural changes are accessible from these studies. In this paper, we have carried out a 70-ps molecular dynamics simulation on a model structure of gramicidin A with channel waters. The backbone of the protein has been analyzed with respect to the orientation of the carbonyl and the amide groups. The results are in conformity with the experimental NMR data. The structure of water and the hydrogen bonding network are also investigated. It is found that the water molecules inside the channel act as a collective chain; whereas the conformation in which all the waters are oriented with the dipoles pointing along the axis of the channel is a preferred one, others are also accessed during the dynamics simulation. A collective coordinate involving the channel waters and some of the hydrogen bonding peptide partners is required to describe the transition of waters from one configuration to the other.     

Fluorescence study on the conformation of a cyclic enkephalin analog in aqueous solution

Certain patients with ovarian germ cell tumors develop a specific antibody reacting with glycoprotein-bound large carbohydrates of murine teratocarcinoma cells. The antigenic determinant was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases abolished the antigenic activity of the large glycan isolated from F9 and OTT6050 cells. Several evidences excluded the possibility that the antigen is blood group B or P1 antigen. These results indicate tumor-associated expression of an unusual alpha-galactosyl residue in human ovarian germ cell tumors.     

Necleoside conformations. 19. Temperature and pH effects on the conformation of guanosine phosphates.

Proton magnetic resonance spectra at 250 MHz were measured as a function of temperature and pH of the three guanosine phosphates. From these data and previously published work the conformational parameters of these compounds were determinated. The phosphate group of Guo-5'-P changes its conformation around the C-O bond and its rotation is relatively slow at 20 degrees. At neutral pD the S conformation is favoured and the N form at acid pD. This conformational change is paralleled by a change in exocyclic rotamer distribution and takes place at the pK of the protonation of the base on N-7. Although correlation appears to exist between the various conformations, notable exceptions exist.     

A simple procedure for the isolation of a major amelogenin polypeptide component.

A simple, reproducible gel-filtration procedure for the isolation of one of the major 'J-Group' polypeptides of the bovine foetal dental-enamel matrix is described. The purified polypeptide was characterized by amino acid analysis, electrophoresis, cleavage with CNBr and N-terminal analyses. The isolated component is shown to be closely similar to an amelogenin component described by other workers.     

Binding to opioid receptors of enkephalin derivatives taking alpha-helical conformation and its dimer.

The opioid receptor binding of [Leu]enkephalin derivatives with extended address segment to the C-terminal was studied. The extension peptide is designed to take an amphiphilic helical structure in order to evaluate effects of helical conformation and membrane affinity of enkephalin moiety of the derivatives on receptor binding. In the delta-receptor-selective binding assay, Tyr-Gly-Gly-Phe-Leu-Lys-Aib-Leu-Aib-OH (1) showed the same affinity as enkephalinamide, whereas in the mu-receptor-selective binding assay, a 7-fold reduction in affinity was observed. On the other hand, Tyr-Gly-Gly-Phe-Leu-(Lys-Aib-Leu-Aib)2-OH (2) showed 51- and 96-fold decreases in affinities for delta- and mu-receptors, respectively, compared with enkephalinamide. The low receptor affinity of derivative 2 is considered due to alpha-helical conformation, which might not be compatible with topological requirements of delta- and mu-receptors. A dimer, Tyr-Gly-Gly-Leu-Phe-(Lys-Aib-Leu-Aib)2-Lys(X)-Aib-OCH3 (X = Tyr-Gly-Gly-Phe-Leu-, (4], showed 2.5- and 3.0-fold increases in affinities respectively for delta- and mu-receptors compared with the monovalent derivative 2, possibly due to cross-linking of neighboring receptors. The Hill plot of the binding of the dimer to bovine brain membranes was composed of two phases, although such a heterogeneity of receptors was not observed in the presence of naloxone or in the binding to NG108-15 cell membranes. These findings indicate the presence of the bivalent-ligand-induced interactions between delta- and mu-receptors in bovine brain membranes.     

Stereochemical analysis of the polypeptide chains secondary structure by Courtauld space-filling models. I. Dipeptide allowed conformations]

For all the 20 natural amino acid residues the conformations, permitted by taking into account steric interactions (a small variation of valent angles was admitted) and interactions with the solvent (water) were singled out.