Approach to studying the structure-activity relationship of native peptides. I. Structural organization of the opioid peptide Tyr-Glu-Gly-Phe-Met-Arg-Gly-Leu and its artificial analogs]

Theoretical conformational analysis was carried out for the octapeptide Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Gly7-Leu8. Possible structure of the opioid peptide under physiological conditions may be described by a set of low-energy conformations belonging to 14 different forms of the backbone. The solution of the "reverse conformational problem" for the opioid peptide enables one to predict the modified amino acid sequences (Ala2, D-Ala2, Ala3, D-Ala3, Ala7, D-Ala7, MeMet5, MeArg6-analogues) 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.     

Structure-functional organization of the secretin molecule. II. Direct conformation problem

Spatial structure of peptide hormone secretin was investigated by the theoretical conformational method. A solution of the "direct conformational problem" for this hormone indicated that the possible structure of the secretin molecule under polar conditions may be described only by two families of low-energy conformations, possessing relatively conformational valid (Thr7-Leu22) and variable (His1-Phe6 and Leu23-Val27NH2) fragments. One of these families is comprised by five twists of the alpha-helix, while the second isoenergetic family possesses two short segments of the alpha-helix, divided by an irregular structure of the tetrapeptide.     

Nuclear magnetic resonance analysis and conformational characterization of a cyclic decapeptide antagonist of gonadotropin-releasing hormone

Two-dimensional proton nuclear magnetic resonance spectroscopy at 500 MHz has been carried out on the cyclic decapeptide antagonist of gonadotropin-releasing hormone: cyclo-(delta 3-Pro1-D-pClPhe2-D-Trp3-Ser4-Tyr5-D-Trp6-NMeLeu7-Arg8- Pro9-beta-Ala 10). The antagonist exists in two slowly interconverting conformations. All data are consistent with the conclusion that one form has all-trans peptide bonds and the other has a cis beta-Ala10-delta3-Pro1 bond. With the use of sequential assignment methods, chemical shift assignments were obtained for all backbone and side-chain protons of both conformational isomers except for the serine and tyrosine hydroxyl groups and the C gamma, C delta, and guanidinium group protons of the arginine. Temperature dependence of spectral parameters and magnitudes of observed nuclear Overhauser effects support the interpretation that both conformers of the antagonist consist of two beta-turns (type II', D-Trp6-NMeLeu7; type II, delta 3-Pro1-D-pClPhe2) connected by extended antiparallel beta-like strands.     

Spatial structure of the cardioactive octapeptide

The spatial structure of the cardioactive octapeptide Pro1-Gln2-Asp3-Pro4-Phe5-Leu6-Arg7-Ile8-NH2 was investigated using the theoretical conformational analysis. The low-energy conformations of the octapeptide molecule were found, the values of dihedral angles of the backbone and side chains of the amino acid residues constituting the peptide were determined, and the energies of intra-and interresidual interactions were estimated. It was shown that the spatial structure of this molecule represent six stable low-energy forms of the main chain.     

Proline-induced constraints in alpha-helices

The disrupting effect of a prolyl residue on an α-helix has been analyzed by means of conformational energy computations. In the preferred, nearly α-helical conformations of Ac-Ala₄-Pro-NHMe and of Ac-Ala₇-Pro-Ala₇-NHMe, only the residue preceding Pro is not α-helical, while all other residues can occur in the α-helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α-helix. Other low-energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near-α-helical A-conformational state (with up to 26° changes of their dihedral angles ? and ψ, and a 48° change in one ω from those of the ideal α-helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α-helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α-helices, 61 have a Pro at or next to their N-terminus, 21 have a Pro next to their C-terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α-helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.     

Conformational analysis of the biologically active cyclic analog of beta-casomorphin H-Tyr-cyclo[D-OrnPheProGly].

A biologically active analog of beta-casomorphin, H-Tyr-cyclo[D-OrnPheProGly], was studied by theoretical conformational analysis. Random sampling was used to search the conformational space of allowed dihedral angles. Among 53 low-energy conformers with different folding of the peptide cyclic moiety, only those were selected which correspond to the low-energy area of the model linear tripeptide Ac-D-AlaAlaPro-NHMe. This peptide was used as the main chain "template" for the D-OrnPheProGly fragment of the studied cyclopeptide molecule. Only 15 conformers were chosen as potentially biologically active structures. The conformational possibilities of the Phe residue were constrained to the combined (A + G) region of the Ala residue phi,psi-map for linear peptides.     

Accessible conformations of melanin-concentrating hormone: a molecular dynamics approach

Molecular dynamics simulations have been used to search for the accessible conformations of the melanin-concentrating hormone (MCH). The studies have been performed on native MCH and two of its peptide fragments, a cyclic MCH(5-14) fragment and a linear MCH(5-14) fragment. An analysis of the molecular dynamics trajectories of the three peptides indicates that two regions of the peptide have characteristic conformational properties that may be important for the biological activity. One is a region around Gly8, which is conformationally mobile, and the other is around Pro13, which shows unusual rigidity. The molecular dynamics simulation results are discussed in terms of backbone structural features like beta turns, side-chain interactions, and orientations of the disulfide bridge. The results of this analysis are used to suggest new analogues that will modify the conformational features of the peptide and further define the conformational requirements for activity. Finally, the results are related to nmr studies of the peptide and reveal agreements between the experimental nuclear Overhauser effect constraints and some of the accessible conformations obtained from the simulation.     

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.     

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.     

An active site of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipose cells

In order to elucidate the active sites of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes, four artificial mutant variants of human growth hormone (hGH) modified in the loop region of amino acid residues 54-74 were prepared in Escherichia coli by site-directed mutagenesis. Although the P59A (replacement of Pro59 with Ala) variant retained almost the same biological- and receptor binding-activity as hGH, the P61A (replacement of Pro61 with Ala) and the P59A-P61A (replacement of both Pro59 and Pro61 with Ala) both exhibited about half the activity, and the delta (62-67) variant (deletion of the residues 62-67) exhibited only about 0.1% the activity of those of intact hGH. The results suggest that Pro61 may be involved in formation of the active conformation of hGH, but Pro59 may not, and that the amino acid residues around 62-67 may be critical for the specific biological features of hGH.     

Peptide inhibitors of E. collagenolyticum bacterial collagenase--effect of N-methylation. Consequences on biological activity and conformational properties.

Peptide inhibitors of E. collagenolyticum bacterial collagenase, HS-CH2-CH2-CO-Pro-Yaa (Yaa = Ala, Leu, Nle), have been N-methylated at the Yaa position. The N-methylation slightly increases the inhibitory potency of the modified peptides as compared to the parent compounds. The conformational effects of the N-methylation have been investigated by both 1H 2D-NMR and molecular mechanics energy minimization. Three low-energy conformers have been predicted for the unmethylated parent compounds (Yaa = Ala, Leu, Nle). They are characterized by the psi value of the central proline residue: psi Pro = 150 degrees (trans' conformation), psi Pro = 70 degrees (C7 conformation) and psi Pro = -50 degrees (cis' conformation). The N-methylation has been found to strongly increase the energy of the C7 conformer and to a less extent the energy of the cis' conformer. This leaves the trans' conformation as the only low-energy conformer. The ROESY experiments have established that both the N-methyl peptides and the parent compounds adopt the same preferred backbone conformation in water solution, i.e. the trans' conformation. Based on these results, the activities of the N-methyl peptides are discussed and a possible conformation of the inhibitor in the bound state is proposed.     

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.     

Study of the spatial structure of des-Gly9-[Arg8]vasopressin by two-dimensional NMR spectroscopy and theoretical conformation analysis

2D 1H-NMR spectra of des-Gly9-[Arg8]vasopressin in dimethylsulfoxide have been taken and the 1H resonances have been assigned. The coupling constants and amide proton temperature coefficients (delta delta/delta T) have been measured and the NOE cross-peaks in the NOESY spectrum have been analyzed. The most essential information on the spatial structure of des-Gly9-[Arg8]vasopressin is extracted from the low delta delta/delta T value for Asn5 amide proton and from the NOE between the Cys1 and Cys6 alpha-protons. A diminished accessibility of the Asn5 NH proton for the solvent is ascribed to the presence of a beta-turn in the fragment 2-5. The distance between the Cys1 and Cys6 C alpha H protons seems to be less than 4 A. These constraints were taken into account in the conformational analysis of the title peptide. The derived set of the low-energy backbone conformations was analyzed against the background of the all available NMR data. The most probable conformation of the cyclic moiety in des-Gly9-[Arg8]vasopressin was found to be the type III beta-turn. The corner positions are occupied by the residues 3, 4, while the residues 1-2 and 5-6 are at the extended sites. Some NMR data indicate that this structure is in a dynamic equilibrium with other minor conformers.     

Cis-trans isomerization of proline in the peptide (his 105-Val 124) of ribonuclease a containing the primary nucleation site

Conformational energy calculations have been carried out to determine the relative stabilities of the C-terminal sequence 105–124 of ribonuclease A, withcis andtrans forms, respectively, of Asn 113-Pro 114. Thecis form of Pro 114 is the one that occurs in the native protein. This peptide contains the sequence 106–118, which, on the basis of both theoretical and experimental studies, is thought to constitute the primary nucleation site for the folding of ribonuclease A. It is shown that both conformations of the isolated peptide (with Pro 114 in thecis andtrans forms, respectively) are of approximately equal stability. Both forms have similar conformations from residues 105–110 and 118–124, while they differ in the bend region involving residues 111–117. Calculations have also been carried out to deduce the possible low-energy paths for the interconversion between thecis andtrans forms of both Pro 114 and Pro 117. It is shown that there are two low-energy paths (with a minimum activation energy of 16.5 kcal/mole) for the interconversion of Pro 114. Attractive nonbonded interaction energies stabilize the transition state on these paths. Only one relatively low-energy path (with an activation energy of 18 kcal/mole) could be found for the isomerization of Pro 117, which occur in thetrans form in the native protein; in this case, allcis forms have significantly higher energy than thetrans form. These calculations thus show that native-like forms for the isolated peptide can exist with Pro 114 in either thecis or thetrans form and that these forms are readily interconvertible.     

Interaction of substance P and its N- and C-terminal fragments with Ca2+: implications for hormone action.

In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1-7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7-11 C-terminal fragment peptide displayed a binding indicative of an 1:1 Ca2+/peptide complex. Ca2+ binding by the hormone and the 7-11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 microM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the molecular basis of the recognition of angiotensin II (AII). NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131.

The high-resolution 3D structure of the octapeptide hormone angiotensin II (AII) in aqueous solution has been obtained by simulated annealing calculations, using high-resolution NMR-derived restraints. After final refinement in explicit water, a family of 13 structures was obtained with a backbone RMSD of 0.73 +/- 0.23 A. AII adopts a fairly compact folded structure, with its C-terminus and N-terminus approaching to within approximately 7.2 A of each other. The side chains of Arg2, Tyr4, Ile5 and His6 are oriented on one side of a plane defined by the peptide backbone, and the Val3 and Pro7 are pointing in opposite directions. The stabilization of the folded conformation can be explained by the stacking of the Val3 side chain with the Pro7 ring and by a hydrophobic cluster formed by the Tyr4, Ile5 and His6 side chains. Comparison between the NMR-derived structure of AII in aqueous solution and the refined crystal structure of the complex of AII with a high-affinity mAb (Fab131) [Garcia, K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T., Amzel, L.M. (1992) Science257, 502-507] provides important quantitative information on two common structural features: (a) a U-shaped structure of the Tyr4-Ile5-His6-Pro7 sequence, which is the most immunogenic epitope of the peptide, with the Asp1 side chain oriented towards the interior of the turn approaching the C-terminus; (b) an Asx-turn-like motif with the side chain aspartate carboxyl group hydrogen-bonded to the main chain NH group of Arg2. It can be concluded that small rearrangements of the epitope 4-7 in the solution structure of AII are required by a mean value of 0.76 +/- 0.03 A for structure alignment and approximately 1.27 +/- 0.02 A for sequence alignment with the X-ray structure of AII bound to the mAb Fab131. These data are interpreted in terms of a biological "nucleus" conformation of the hormone in solution, which requires a limited number of structural rearrangements for receptor-antigen recognition and binding.     

Conformational study of the neurotensin and substance P fragments: Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro

The conformational behaviour of the basic hydrophilic Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro peptides, neurotensin (NT) and Substance P fragments, has been taken up by semi-empirical calculations. The presence of two Pro residues prevents these peptides from giving any folded structure (alpha helix, beta turn . . .). In both peptides the most stable conformations are essentially relative to more or less stretched structures; structures involving one or more residues in a gamma turn form are often encountered in Pro-Arg-Arg-Pro peptide while mixed structures involving residues in very different conformations are found for the Arg-Pro-Lys-Pro-peptide. In both peptides, positively charged Lys and Arg side-chains most often point in opposite directions. The Pro-Arg-Arg-Pro peptide is part of the active NT (7-13) fragment where both Arg residues are necessary to the activity. A tentative study shows that the hydrophilic tetrapeptide induces NT (7-13) stretched conformations.     

Morphiceptin and beta-casomorphin-5 analogues containing a reduced peptide bond: selective mu-receptor agonists and a novel mu antagonist, H-Tyr-Pro psi (CH2-NH)Phe-Pro-Gly-OH.

In order to prevent enzymatic degradation of beta-casomorphin-5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2-3 and 3-4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin-bound peptide amines with Boc-amino acid aldehydes (Boc: tert-butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (less than 3%) in the crude product. Instead, the phenomena could be attributed to slow cis/trans isomerizations originating from the Xxx-Pro bonds in the sequence. The presence of different conformational isomers was also established by 1H-nmr spectroscopy in DMSO-d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the mu receptor, and very low binding to the delta receptor. While the Phe 3 psi(CH2-N)Pro4 analogues (3) and (5) displayed agonist activity, the Pro 2 psi(CH2-NH)Phe3 modified analogue (2) showed antagonist activity comparable to D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2.     

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.