Conformational analysis of the biologically active RGD-containing anti-adhesive peptide cyclo(ArgGlyAspPhe-D-val)

Using theoretical conformational analysis, the RGD-peptide with anti-adhesive activity cyclo(ArgGlyAspPhe-D-Val) was studied. Random sampling was used to search the conformational space of the allowed torsional angles of the main chain of the molecule. Among 900 stable conformers with different folding of the cyclic moiety of the peptide, only those were selected which corresponded to low-energy conformers of the model linear tripeptide AcAlaGlyAspNHMe. This peptide served as the main chain template of the RGD-fragment of the studied cyclopeptide molecule. Of 36 selected cyclopeptide conformers with potential biological activity, only a few contain stable intramolecular hydrogen bonds. It was supposed that a biologically active conformer of the cyclopeptide molecule exists in solution among other conformers, but not necessarily as the major component of the equilibrium mixture.     

A conformational analysis of biologically active RGD-containing cyclopentapeptides

The theoretical conformational analysis of the biologically active RGD-containing pentapeptide cyclo(-Arg-Gly-Asp-Phe-DVal-), an inhibitor of laminin P1 interaction with its receptor, was performed. The space of permissible torsional angles of the backbone of the molecule was studied by the Monte Carlo method. From the large number of predicted low-energy conformers with various packings of the cyclic moiety of this pentapeptide, only those were selected that corresponded to stable structures of the model linear tripeptide Ac-Ala-Gly-Asp-NHMe. This peptide simulated the spatial possibilities of the backbones of RGD-containing fragments of laminin, vitronectin, and fibronectin. We selected several dozen structures that may be potential biologically active conformers, but only a few of them were capable of forming stable intramolecular hydrogen bonds. We assumed that a biologically active conformer of cyclo(-Arg-Gly-Asp-Phe-DVal-) can be present in significant amounts in an equilibrium mixture in solution along with other conformers without necessarily dominating among them.     

Conformational analysis of biologically active RGD-containing cyclopentapeptides

The theoretical conformational analysis of the biologically active RGD-containing pentapeptide cyclo(-Arg-Gly-Asp-Phe-DVal-), an inhibitor of laminin P1 interaction with its receptor, was performed. The space of permissible torsional angles of the backbone of the molecule was studied by the Monte Carlo method. From the large number of predicted low-energy conformers with various packings of the cyclic moiety of this pentapeptide, only those were selected that corresponded to stable structures of the model linear tripeptide Ac-Ala-Gly-Asp-NHMe. This peptide simulated the spatial possibilities of the backbones of RGD-containing fragments of laminin, vitronectin, and fibronectin. We selected several dozen structures that may be potential biologically active conformers, but only a few of them were capable of forming stable intramolecular hydrogen bonds. We assumed that a biologically active conformer of cyclo(-Arg-Gly-Asp-Phe-DVal-) can be present in significant amounts in an equilibrium mixture in solution along with other conformers without necessarily dominating among them.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

Theoretical conformational analysis of a μ-selective cyclic opioid peptide analog

The allowed conformations of the μ-receptor-selective cyclic opioid peptide analog were determined using a grid search through the entire conformational space. Energy minimization of the 13-membered ring structure lacking the exocyclic Tyr¹ residue and the Phe³ side chain using the molecular mechanics program Maximin resulted in only four low-energy conformations. These four ring structures served as templates for a further energy minimization study with the Tyr¹ residue and Phe³ side chain added to the molecule. The results indicated that the Tyr¹ and Phe³ side chains enjoy considerable orientational freedom, but nevertheless, only a limited number of low-energy side-chain configurations were found. The obtained low-energy conformers are discussed in relation to various proposed models of the bioactive conformation of enkephalins and morphiceptin.     

Conformation-activity relationships of cyclic dermorphin analogues

A theoretical conformational analysis (molecular mechanics study) of nine cyclic tetrapeptides, structurally related to the highly mu-receptor-selective dermorphin analogue H-Tyr-D-Orn-Phe-Asp-NH2, was performed. These compounds display considerable diversity in their mu-receptor affinity and selectivity. A systematic search and subsequent energy minimization in absence of the exocyclic Tyr1 residue and Phe3 side chain revealed the constrained nature of the 11-13-membered ring structures contained in these analogues. No more than four low-energy conformers (within 2 kcal/mol of the lowest energy conformation) were found in each case. After attachment of the Tyr1 moiety and Phe3 side chain to the "bare" low-energy ring structures, a systematic search and energy minimization of these exocyclic moieties resulted in a limited number of low-energy conformational families for all compounds. Five analogues with high mu-receptor affinity--H-Tyr-D-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-Phe-D-Asp-NH2, H-Tyr-D-Asp-Phe-Orn-NH2, H-Tyr-D-Asp-Phe-A2bu-NH2 (A2 bu: alpha, gamma-diaminobutyric acid) and H-Tyr-D-Cys-Phe-Cys-NH2--all showed a tilted stacking interaction between the Tyr1 and Phe3 aromatic rings in the lowest or second lowest energy conformation found. The same kind of stacking was not possible in low-energy conformers of the four analogues with poor affinity for the mu-receptor [H-Tyr-L-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-D-Phe-Asp-NH2, H-Tyr-D-Orn-Phe(NMe)-Asp-NH2 [Phe(NMe): N alpha-methylphenylalanine], and H-Tyr-D-Orn-Phg-Asp-NH2 (Phg: phenylglycine)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Ab initio modeling of small, medium, and large loops in proteins.

This study presents different procedures for ab initio modeling of peptide loops of different sizes in proteins. Small loops (up to 8--12 residues) were generated by a straightforward procedure with subsequent "averaging" over all the low-energy conformers obtained. The averaged conformer fairly represents the entire set of low-energy conformers, root mean square deviation (RMSD) values being from 1.01 A for a 4-residue loop to 1.94 A for an 8-residue loop. Three-dimensional (3D) structures for several medium loops (20--30 residues) and for two large loops (54 and 61 residues) were predicted using residue-residue contact matrices divided into variable parts corresponding to the loops, and into a constant part corresponding to the known core of the protein. For each medium loop, a very limited number of sterically reasonable C(alpha) traces (from 1 to 3) was found; RMSD values ranged from 2.4 to 5.9 A. Single C(alpha) traces predicted for each of the large loops possessed RMSD values of 4.5 A. Generally, ab initio loop modeling presented in this work combines elements of computational procedures developed both for protein folding and for peptide conformational analysis.     

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.     

Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.     

CD spectrum and conformational distribution of cyclotuftsin in solution

CD spectra for low-energy conformations of the tuftsin cycloanalogue, , were calculated. A theoretical spectrum obtained as the weighted average of calculated spectra for individual peptide backbone conformers is qualitatively consistent with an experimental CD spectrum in aqueous solution. The conformational distribution allows one to achieve agreement between calculated and experimental values of structural parameters of the cyclotuftsin molecule investigated by NMR spectroscopy.

CD spectrum calculation Theoretical conformational analysis Tuftsin cycloanalog Peptide conformation     

Conformational features responsible for binding of cyclic analogues of enkephalin to opioid receptors. I. Low-energy peptide backbone conformers of analogues containing Phe4

Low-energy peptide backbone conformers were found by means of energy calculation for several cyclic analogues of enkephalin in an attempt to assess models for receptor-bound conformations for opioid receptors of the mu- and delta-types. They included [D-Cys2, L-Cys5]- and [D-Cys2, D-Cys5]-enkephalinamides showing moderate preference for mu-receptors, the delta-selective compounds [D-Pen2, L-Pen5] and [D-Pen2, D-Pen5]-enkephalins and Tyr-D-Lys-Gly-Phe- analogue possessing very high affinity to receptors of the mu-type. The low-energy conformers obtained for these analogues were in good agreement with the results of calculations by other authors and with experimental evidence. All of the analogues contain a Phe residue in position 4 of the peptide chain which facilitates the eventual search for geometrical similarity between the low-energy backbone conformers of different analogues in question.     

Role of proximal His93 in nitric oxide binding to metmyoglobin. Application of continuum solvation in Monte Carlo protein simulations.

Monte Carlo protein simulations with continuum solvation were used to explore the conformational mobility of NO within the active site of metmyoglobin. To the best of our knowledge this is the first application of a continuum solvation model for exploring protein binding sites. The usefulness of the Monte Carlo conformational analysis was demonstrated in comparative molecular dynamics simulations. Analysis of conformer populations revealed that Monte Carlo conformational analysis is more effective in mapping the relevant region of the potential surface. Distribution of low-energy conformers obtained for the metmyoglobin-NO complex was found to depend on the orientation of proximal His93. Different charge distributions corresponding to the two experimentally verified possible torsions of this proximal residue result in strong binding of NO or its release to a nearby hydrophobic trap. Conformer populations obtained by Monte Carlo conformational analysis were grouped into three main families: one, with the NO directly bound to the iron, appears when the CA-CB-CG-CD2 torsion of His93 is at its ligand binding value (-113 degrees); and two conformers exist where NO is trapped in a nearby hydrophobic pocket, the same cavity that was determined to be the geminate trap of CO in ferrous Mb as a result of the torsional flip of His93 to its ligand releasing state (-125 degrees). Based on this analysis, we suggest that the electrostatic rearrangement coupled to the conformational fluctuation of the proximal His leads to the geminate trapping of the ligand. Conformational rearrangement of the proximal side would provide the possibility of rebinding of the ligand to Fe.     

Structural aspects of Ca2+ binding by acyclic peptides: low-energy conformational domains and molecular dynamics of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide.

We have applied random-search, energy minimization and molecular dynamics simulations to investigate the structural aspects of the interaction of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide with Ca2+. Spectral data on related peptides had suggested that the beta-turn conformation might be a prerequisite for the binding of cation ion by such short linear peptides. In order to relate the conformational characteristics with the Ca(2+)-binding affinities of these peptides, the molecular events involved in cation binding need to be understood. We have addressed this problem in this study by using a systematic approach that involved the following steps. First, a random search technique was used to generate a large population of conformers for the free peptide in the absence of Ca2+. Next, the energies of these conformers were computed. Conformations with energies within 4 kcal/mol of the global minimum were analysed and found to fall into four main groups characterized by the presence of different types of hydrogen-bonded structures including single and consecutive beta-turns. The energies for interconversion of conformers from one group to another were computed and found to be relatively small (< 10 kcal/mol). Finally, molecular dynamics of the peptide at 300K in the presence of Ca2+ were used to simulate the cation binding process. Starting points for these simulations were generated by placing the ion in the vicinity of two molecules of the peptide. The simulation results showed that the conformers with two consecutive beta-turns led to the formation of a stable 2:1 (peptide:Ca2+) sandwich complex in agreement with earlier experimental observations on similar linear peptides. While the starting conformation of the peptide in the consecutive beta-turn structure allowed for the proper orientation of three carbonyl oxygen atoms for chelation to the metal ion, the dynamics of complex formation rearranged the peptide structure substantially, leading to the formation of an 8-coordinated Ca2+ complex in a dodecahedral spatial arrangement. Thus, based on the energetics of the structures and processes involved, the present study demonstrates that: a) peptide-Ca2+ complex formation is initiated by conformers adopting consecutive beta-turn structures which subsequently go over to a significantly different conformation found in the complex; and, b) The facile interconversion between the low-energy conformers in the different groups would help shift the equilibrium population towards the consecutive beta-turn structure during the complex formation.     

Structural effects of O-glycosylation on a 15-residue peptide from the mucin (MUC1) core protein

To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, the 15-residue peptide PPAHGVTSAPDTRPA (PPA15) from the MUC1 protein core and its analogue PPA15(T7), glycosylated with alpha-N-acetylgalactosamine on Thr7, were prepared and investigated by NMR spectroscopy. The peptide contains both the GVTSAP sequence, which is an effective substrate for GalNAc-T1 and -T3 transferases, and the PDTRP fragment, which is a well-known immunodominant epitope recognized by several anti-MUC1 monoclonal antibodies. Useful structural results were obtained in water upon decreasing the temperature to 5-10 degrees C. The sugar attachment slightly affected the conformational equilibrium of the peptide backbone near the glycosylated Thr7 residue. The clustering of low-energy conformations for both PPA15 and PPA15(T7) within the GVTSAP and APDTRP fragments revealed structural similarities between glycosylated and nonglycosylated peptides. For the GVTSAP region, minor but distinct clusters formed by either PPA15 or PPA15(T7) conformers showed distinct structural propensities of the peptide backbone specific for either the nonglycosylated or the glycosylated peptide. The peptide backbone of the APDTRP fragment, which is a well-known immunodominant region, resembled an S-shaped bend. A similar structural motif was found in the GVTSAP fragment. The S-shaped structure of the peptide backbone is formed by consecutive inverse gamma-turn conformations partially stabilized by hydrogen bonding. A comparison of the solution structure of the APDTRP fragment with a crystal structure of the MUC1 peptide antigen bound to the breast tumor-specific antibody SM3 demonstrated significant structural similarities in the general shape.     

Theoretical conformational analysis of a encephalitogenic peptide molecule and a study of the structure-activity relationship in a series of its analogs

Semi-empirical energy calculations are used to determine all low-energy conformations of Trp-containing fragment 113-121 of myelin basic protein (experimental allergic encephalomyelitis inducing peptide). The computed conformations are compared with the results of physico-chemical experiments and data on biological testing of the encephalitogenic peptide analogs. The three computed structures are shown to be in a good agreement with the available experimental evidence. However, additional information is required to predict "biologically active" conformation of encephalitogenic peptide.     

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) and [D -Phe⁷]α-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly¹⁰ residue, as substitutions for L -Ala, D -Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH(4–11) and α-MSH(5–11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly¹⁰ residue with L -Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp “message” sequence within the sequences of α-MSH and [D -Phe⁷]α-MSH. In such conformations the aromatic moieties of the side chains of the His⁶, L/D -Phe⁷, and Trp⁹ residues form a continuous hydrophobic “surface,” presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D -Phe⁷]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 155–167, 1998     

A theoretical study of pentacyclo-undecane cage peptides of the type [Ac-X-Y-NHMe].

The conformational preferences of peptides of the type, Ac-X-Y-NHMe, where X and Y = Ala, cage and Pro, were studied by means of computational techniques within the framework of a molecular mechanics approach. For each of the eight peptide analogues, extensive conformational searches were carried out using molecular dynamics (MD) and simulated annealing (SA) protocols in an iterative fashion. Both results are in good agreement and complement each other. The conformational search indicates that the cage residue restricts the conformational freedom of the dipeptide considerably in comparison with the other model residues used. This study revealed that proline exhibits a greater tendency in promoting reverse-turn characteristics in comparison to the cage peptides, which show promising beta-turn characteristics. It was also found that 300-500 K is not sufficient to overcome rotational barriers for cage peptides. In all cases, the low-energy conformers have a tendency to form bent structures.     

Structuro-functional organization of delta sleep-inducing peptide

Theoretical conformational analysis was carried out for a nonapeptide hormone (delta sleep-inducing peptide). Possible structure of the neuropeptide under physiological conditions may be described by a set of low-energy conformations belonging to nine different forms of the backbone. A solution of the "reverse conformational problem" for delta sleep inducing peptide enables one to predict modified amino acid sequences (D-Ala3-, Pro4-, Pro6-, Pro7, and Tyr7-analogs), which may assume one of the low-energy states of the native hormone. The influence of the solute was not taken into account in our calculations.