The solution conformation Ac-Pen-Arg-Gly-Asp-Cys-OH,a potent fibrinogen receptor antagonist

The solution conformation of , a potent fibrinogen receptor antagonist, was characterized in DMSO-d₆ by the combination of nmr and molecular modeling. The conformational space available to the peptide was explored using a distance geometry algorithm with distance constraints derived from ¹H-nmr spectra. The dynamics of the peptide were examined by relaxation time measurements and low temperature studies. The results from the low temperature studies suggest that the peptide backbone does not exist in a single, well-defined conformation but undergoes exchange between multiple conformers. This result is consistent with the inability to find a single structure that satisfies all the nmr-derived constraints. The constraints could only be satisfied by considering pairs of conformers to represent the experimental data. The low energy conformers comprise type II′ or type V β-turns with distinct side-chain directionality. The Arg-Gly-Asp portion of the ring is flexible and can be described by amide-plane rotations of the Arg-Gly and Gly-Asp peptide bonds. Although some backbone flexibility is evident, the incorporation of β,β-dimethyl cysteine imparted greater conformational rigidity as compared to the previously studied cyclic pentapeptide, . © 1993 John Wiley & Sons, Inc.     

Conformational study of cyclo[D-Trp-D-Asp-Pro-D-Val-Leu], an endothelin-A receptor-selective antagonist.

The conformation of cyclo[D-Trp-D-Asp-Pro-D-Val-Leu], (BQ123), an endothelin-A receptor-selective antagonist, has been studied in 20% acetonitrile in water by CD and NMR spectroscopy. CD studies showed the peptide adopted a similar, constrained conformation in both water alone and 20% acetonitrile in water. NMR spectra showed the proline residue to be in the trans conformation and 2 of the NH protons to exchange slowly with the solvent, indicating hydrogen bonding. Structural constraints derived from the NMR spectra were used to define the conformation in molecular dynamics simulations. A single backbone conformation is observed for the cycle, comprising a beta type II turn and a gamma' turn.     

Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.     

Photomodulation of conformational states. IV. Integrin-binding RGD-peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

In previous studies we have investigated octapeptides backbone-cyclized by (4-amino)phenyl azobenzoic acid (APB) or (4-aminomethyl)phenylazobenzoic acid (AMPB) and containing the active-site sequence Cys-Ala-Thr-Cys-Asp from the thioredoxin reductase. The conformational and redox properties of these peptides were strongly dependent on the isomeric state of the azobenzene chromophore. Using the same approach we were successful in constructing photoresponsive ligands for alphavbeta3 integrin containing the Arg-Gly-Asp (RGD) sequence as binding motif. For achieving maximal conformational restriction of the peptide a reduced ring size compared to our previous azobenzene peptides was employed in the cyclic peptide c[Asp-D-Phe-Val-AMPB-Lys-Ala-Arg-Gly-]. Conformational properties of the trans and cis isomers of this peptide in solution were investigated by CD and NMR and were found to differ markedly from the thioredoxin derived azobenzene peptides. In a second peptide, c[Asp-D-Phe-Val-Lys-AMPB-Ala-Arg-Gly-], shifting the position of the chromophore lead to a marked decrease in affinity. With the availability of the x-ray structure of a cyclic RGD-pentapeptide bound to alphavbeta3 integrin (PDB entry 1L5G) modeling of possible bound conformations for trans and cis isomers of both azobenzene peptides was possible. Notably, both peptides in either isomeric form share the same overall conformation in the bound state according to our molecular dynamics simulations.     

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.     

Nearest-neighbor parameters for G.U mismatches: [formula; see text] is destabilizing in the contexts [formula; see text] and [formula; see text] but stabilizing in [formula; see text].

Thermodynamic parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides containing G.U mismatches. The results are used to determine nearest-neighbor parameters for helix propagation by G.U mismatches. Surprisingly, the [formula; see text] nearest-neighbor free energy increment in unfavorable in the contexts [formula; see text], and [formula; see text] but favorable in the context [formula; see text]. This is a non-nearest-neighbor effect. In contrast, the [formula; see text] free energy increment is favorable and independent of context. Circular dichroism and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G.U mismatches have two slowly exchanging imino protons. The imino resonances for the G.U mismatches in GGAGUUCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temperature than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G.U mismatches in GGAUGUCC remain sharp at high temperature. The improved parameters for G.U mismatches should improve predictions of RNA structure from sequence.     

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.     

Solution Conformation of [D-Pen2,D-Pen5]enkephalin in Water: A NMR and Molecular Dynamics Study

The solution conformation of [D -Pen²,D -Pen⁵] enkephalin (DPDPE), a highly potent δ-selective opioid agonist, was examined by means of NMR, molecular mechanics and molecular dynamics methods. The structural information in the solvent water was obtained employing one- and two-dimensional methods of ¹H and ¹³C-NMR spectroscopy. Based on the distance geometry technique using the ROE data as input, 400 conformers were obtained and considered in the structure analysis. Alternatively, about 2000 conformers were stochastically generated and related to the NMR data after energy minimization. The structure analysis provides one conformer in agreement with all NMR data, which belongs to the lowest energy conformation group. This structure may serve as a reference conformer for DPDPE analogues synthesized with the aim of activity increase.     

Structure refinement of a cyclic peptide from two-dimensional NMR data and molecular modeling

The conformational and dynamic properties of a cyclic peptide designed to inhibit human renin have been examined by using NMR and molecular modeling. From a quantitative analysis of a series of two-dimensional NOE data sets, proton-proton distances were calculated. Several different methods were explored and compared to incorporate these distance constraints as well as those derived from vicinal spin-spin coupling constants into computer-generated three-dimensional structures. These methods included interactive manual manipulation of the structures to fit the NMR-determined distance constraints, distance geometry, constrained energy minimizations, and constrained molecular dynamics. The advantages and disadvantages of the methods are discussed. In addition, to gain insight into the conformations accessible to the cyclic peptide and the relative flexibility of the different parts of the molecule, molecular dynamics calculations were performed at three different temperatures. Average interproton distances and dihedral angles were obtained from the structures generated in the dynamics trajectories and compared to those obtained from the NMR experiments. Despite the four methylene groups and ether linkage contained in the cyclic portion of the peptide, our NMR results indicated a preferred conformation for the macrocyclic ring of the peptide and supported the presence of a cis Phe-Ala peptide bond. In contrast, both the molecular dynamics and NMR data indicated a considerable amount of flexibility for the remaining noncyclic portion of the molecule. These results are used to propose an explanation for the cyclic peptide's inability to inhibit human renin.     

Conformational studies of human islet amyloid peptide using molecular dynamics and simulated annealing methods

Molecular dynamics simulations and simulated annealing in vacuum, model aqueous solution, and simulated membrane were used to analyze the conformational preferences of a segment spanning 20–29 residues of human islet amyloid polypeptide, [referred to as IAPP^H(20–29)]. Molecular dynamics simulations were conducted at 300 K on IAPP^H(20–29). The minimum energy conformers obtained in model aqueous solution and vacuum exhibited similar structures. Even in the absence of any constraints on peptide bonds, trans conformation was preferred consistently by all the peptide bonds. Analysis of the minimum energy conformers indicated that IAPP^H(20–29) showed a strong preference for turn structures in all the environments. These turn structures were stabilized by the formation of hydrogen bonds between the backbone amide and carbonyl groups. A good agreement was found between the results obtained from the molecular dynamics simulation and solid-state nmr experimental studies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 9–20, 1998     

Conformational analysis of receptor selective tachykinin analogs: senktide and septide.

The conformational behavior in solution of two receptor selective tachykinin agonists, senktide (succinyl-D-F-MeF-G-L-M-NH2) and septide (pQ-F-F-P-L-M-NH2) is described. Two dimensional cross relaxation NMR spectroscopy is used together with coupling constant data to obtain interproton distance constraints. These results are used in conjunction with semi-empirical energy computations to indicate favorable conformations. Senktide is found to have a high degree of conformational order which is attributed to rotational restriction associated with the N-methylation of phenylalanine. The lowest energy conformation in accord with the experimental interproton distances contains a beta-turn. Interproton distances indicate that septide exists as a random coil or in an extended chain conformation. Energy computations suggest that septide is primarily an extended chain with internal reorientation restricted by the proline residue. These results may be related to the selectivity of these peptides for different receptors, in that the analogs, with conformations more stable than tachykinins, are more receptor selective.     

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.     

Novel mu-selective Met-enkephalinamide analogs with antagonist activity. Synthesis, receptor binding, analgesic properties and conformational studies

Four novel mu-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, beta-methyl-m-Tyr, N-phenethyl-m-Tyr and alpha, beta-dimethyl-m-Tyr into D-Ala2-Met5-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala2-Met5-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a beta-CH3 group to the m-Tyr enhances mu-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high mu-receptor affinity examined have a common energy accessible B'II 2-3 turn conformation similar to that previously identified for high mu-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.     

Synthesis, activity on NK-3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16.

Two analogs of a tachykinin family peptides - scyliorhinin II (ScyII): [Aib(16)]ScyII and [Sar(16)]ScyII were synthesized by the solid-phase method using Fmoc chemistry. Conformational studies in water and DMSO-d(6) on these peptides were performed using a combination of two-dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar(16)]ScyII in water appeared to be more flexible, especially in the C-terminal fragment. A better defined structure for this analog was obtained in DMSO-d(6), in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 3(10)-helix in the N-terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib(16)]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 3(10)-helix in the 13-18 fragment. A common structural motif was found in conformationally constrained Cys(7)-Cys(13) fragment, which resembles the Greek letter 'omega'. The differences in the solution structure of the C-terminal fragment of the peptides studied are responsible for their specificity. [Aib(16)]ScyII showed 25% the agonistic activity of selective NK-3 agonist - senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar(16)]ScyII appeared to be a full agonist of NK-3 tachykinin receptor.     

Preferred conformations of endogenous cannabinoid ligand anandamide

Anandamide (arachidonyl-ethanolamide, AEA) is an important endogenous cannabinoid ligand isolated from porcine brain. AEA has a flexible molecular structure with a series of four non-conjugated double bonds, a hydrophobic alkyl chain, and a carboxyamide head group. It is known that AEA binds to cannabinoid receptor and induces cannabimimetic activity. However, questions still remain about the three-dimensional arrangement of the pharmacophoric groups of AEA that facilitate its interaction with cannabinoid receptor, a member of transmembrane G-protein coupled receptors (GPCRs). Such information is of critical importance for the design of novel analogs of potential therapeutic values. In the present studies, we developed a combined approach of 2D high-resolution NMR and computer modeling to investigate conformational features of AEA in solution. The developed method and experimental data is then applied to study the structural properties of AEA in a membrane-like environment that will be reported elsewhere. In addition to the measured NOEs, the dihedral angle constraints were for the first time being used as experimentally-determined structural constraints for performing molecular dynamics simulations to refine the NMR-determined AEA conformations. Our results showed that AEA prefers an extended pseudo-helical conformation in solution with two oxygen atoms pointing towards the same side and a straight pentyl chain, which was an averaged conformation observed on the basis of NMR time scale. The results were correlated to the computer predicted AEA models reported by others. The established NMR-based computational approach provides an alternative way to explore further the detailed conformational properties of AEA that encodes important pharmacophoric and conformational information regarding the activation of cannabinoid receptors.     

Structural characterization of cyclic kallidin analogues in DMSO by nuclear magnetic resonance and molecular dynamics.

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys(0)]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr(6)]-KL (YKL), [Trp(6)]-KL (WKL), cyclo-([Tyr(6)]-KL) (YCKL) and cyclo-([Trp(6)]-KL) (WCKL).The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL.The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds.The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed.     

Evidence for a C-terminal structural motif in gastrin and its bioactive fragments in membrane mimetic media

The conformational preferences of human little gastrin, [Nle(15)] gastrin-17, and its short analogues, gastrin-4 and [beta-Ala(1)] gastrin-5, which include the C-terminal tetrapeptide sequence Trp-Met-Asp-Phe-NH(2) crucial for gastrin bioactivity, were determined by NMR spectroscopy in aqueous solutions of zwitterionic dodecylphosphocholine micelles. Backbone HN chemical shift temperature variance, Halpha chemical shift deviations and complex non-sequential NOE patterns pointed to the C-terminal of [Nle(15)] gastrin-17 adopting an ordered conformation. Distance geometry calculations and NOE-restrained molecular dynamics simulations in membrane mimetic solvent boxes of decane and water indicated the C-terminal tetrapeptide sequence of all three peptides adopted a similar, well defined structure, with a general type IV beta-turn observed for all three peptides. The conformation of [Nle(15)] gastrin-17 consisted of two short helices between Leu(5)-Glu(9) and Ala(11)-Trp(14), with the one helix terminating in a type I beta-turn spanning Gly(13)-Asp(16). The experimental evidence and conformational characteristics of the three peptides in micellar media support a membrane-associated mechanism of receptor recognition and activation for the gastrin hormone family and furthermore point to a possible biologically relevant structural motif for gastrin activity.     

Molecular mechanics and dynamics of an abasic frameshift in DNA and comparison to NMR data

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide: [sequence; see text] where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2' endo or C3' endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4. In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium. Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.     

Role of substrate conformational features in the stereospecificity of aromatase

Hydroxylation of 19-hydroxyandrost-4-ene-3,17-dione (19OHA) by aromatase occurs at the 19-pro-R hydrogen, suggesting that the C19 group has a preferred conformation in the enzyme active site. X-ray crystallographic studies have led to a postulate that the steroid plays a role in determining this conformation. In an effort to quantitate the steroid's role, we estimated conformational constraints about the C10-C19 bond of 19OHA using molecular mechanics calculations. Rotational barriers less than or equal to 6 kcal/mol and energy differences between conformers less than or equal to 1 kcal/mol were found. We perturbed these conformational constraints by preparing an altered substrate, 19-hydroxyandrosta-4,6-diene-3,17-dione (19OHAD). The stereospecificity of aromatization for 19OHA and 19OHAD was found to be the same. Thus, theoretical and experimental approaches both indicate that conformational constraints intrinsic to 19OHA cannot be a major determinant in the sterospecificity of its oxidation by aromatase.