NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

VCD spectroscopic and molecular dynamics analysis of the Trp-cage miniprotein TC5b

TC5b is a 20 residue polypeptide notable for its compact tertiary structure, a rarity for a short peptide. This structure is due to the "Trp-cage" motif, an association of aromatic, Pro, and Gly residues. The structure of TC5b has been fully characterized by NMR and electronic circular dichroism (ECD) studies, but has never been studied with vibrational circular dichroism (VCD) spectroscopy, which may reveal finer structure. In this study, we examine the VCD spectra of TC5b to characterize the spectroscopic signature of the peptide and its comprising structural elements. TC5b exhibited a negative-positive-negative triplet which is associated with alpha-helical structure in deuterated solvents but also signs of a polyproline II (PPII) helix in the amide I' region. Detection of this element was complicated by the aforementioned triplet form, as well as by an upfrequency shift in PPII helical elements due to the use of the deuterated organic solvents DMSO-d(6) and TFE-d(1). Nevertheless, while ECD spectra showed only alpha-helical structure for TC5b, VCD spectroscopy revealed a more complex structure which was in agreement with NMR results. VCD spectroscopy also showed a rapid conformational change of the peptide at temperatures above 35 degrees C in D(2)O and in aqueous solvent with greater than 75% DMSO-d(6) content. Molecular dynamics (MD) simulations to investigate this latter effect of DMSO-d(6) on TC5b were conducted in DMSO and 50% (v/v) DMSO in H(2)O. In DMSO unfolding of the peptide was rapid while in 50% (v/v) DMSO in H(2)O the unfolding was more gradual.     

Characterization of secondary amide peptide bond isomerization: thermodynamics and kinetics from 2D NMR spectroscopy

Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH(2) , Ac-I-G-G-NH(2) , Ac-I-G-G-N-NH(2) and its cyclic form: I-G-G-N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13-0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4-9 × 10(-3) s(-1) . Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints.     

Conformational studies of vasopressin analogues modified with N-methylphenylalanine enantiomers in dimethyl sulfoxide solution

The conformations of [Arg8]vasopressin (AVP) analogues substituted at positions 2 and 3 with N-methylphenylalanine (MePhe) enantiomers were earlier investigated by using nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. A comparison of the results obtained in H2O/D2O (9:1) and DMSO-d6 has shown the structures in the first solution to be more flexible than those in DMSO-d6. This is manifested by a higher percentage of minor conformations in H2O/D2O. The largest differences between the NMR spectra in both solvents were noticed for [MePhe2, D-MePhe3]AVP (II) and [D-Cys1,MePhe2,D-MePhe3]AVP (III). Namely, in the ROESY spectra in aqueous solution, the cis/trans isomerization between MePhe2-DMePhe3 and D-Cys1-MePhe2 for II and III, respectively, is observed, while in DMSO-d6, the appropriate cross peaks indicate isomerization across the Cys6-Pro7 peptide bond. In the case of the remaining peptides, the position of cis/trans isomerization is the same in aqueous solution and in dimethyl sulfoxide. [D-MePhe2,MePhe3]AVP (V) displays low antiuterotonic and antipressor activities, while [D-MePhe2,)]AVP (IV) is a weak but selective blocker of oxytocin (OT) receptors in the uterus. The former shows similar conformational preferences as another antagonist of V1a and OT receptors-namely, [Acc2,D-Arg8]VP (Acc: 1-aminocyclohexane-1-carboxylic acid)-investigated by us. In the case of IV, the cis peptide bond between residues at positions 2 and 3 might be the reason for selectivity.     

Position effect of cross-strand side-chain interactions on beta-hairpin formation

Previous conformational analysis of 10-residue linear peptides enabled us to identify some cross-strand side-chain interactions that stabilize beta-hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N- and C-termini of these 10-residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on beta-hairpin formation, we have designed two 15-residue beta-hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15-residue peptides suggest that the influence of stabilizing factors for beta-hairpin formation, in particular, cross-strand side-chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in beta-hairpin folding.     

Conformational analysis of a 12-residue analogue of mastoparan and of mastoparan X.

We have investigated the conformational properties of a truncated analogue of mastoparan and of mastoparan X, both peptides from wasp venom. The electrostatically driven Monte Carlo method was used to explore the conformational space of these short peptides. The initial conformations used in this study, mainly random ones, led to alpha-helical conformations. The alpha-helical conformations thus found exhibit an amphipathic character. These results are in accord with experimental data from NMR and CD spectroscopy.     

Solution conformational study of Scyliorhinin I analogues with conformational constraints by two-dimensional NMR and theoretical conformational analysis.

Two analogues of Scyliorhinin I (Scyl), a tachykinin with N-MeLeu in position 8 and a 1,5-disubstituted tetrazole ring between positions 7 and 8, introduced in order to generate local conformational constraints, were synthesized using the solid-phase method. Conformational studies in water and DMSO-d6 were performed on these peptides using a combination of the two-dimensional NMR technique and theoretical conformational analysis. The algorithm of conformational search consisted of the following three stages: (i) extensive global conformational analysis in order to find all low-energy conformations; (ii) calculation of the NOE effects and vicinal coupling constants for each of the low energy conformations; (iii) determining the statistical weights of these conformations by means of a nonlinear least-squares procedure, in order to obtain the best fit of the averaged simulated spectrum to the experimental one. In both solvents the three-dimensional structure of the analogues studied can be interpreted only in terms of an ensemble of multiple conformations. For [MeLeu8]Scyl, the C-terminal 6-10 fragment adopts more rigid structure than the N-terminal one. In the case of the analogue with the tetrazole ring in DMSO-d6 the three-dimensional structure is characterized by two dominant conformers with similar geometry of their backbones. They superimpose especially well (RMSD = 0.28 A) in the 6-9 fragments. All conformers calculated in both solvents superimpose in their C-terminal fragments much better than those of the first analogue. The results obtained indicate that the introduction of the tetrazole ring into the Scyl molecule rigidifies its structure significantly more than that of MeLeu.     

Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3.

This paper reports a detailed conformational characterization in solution by 1H-NMR in H2O and DMSO-d6 and molecular modeling simulations of cyclic peptides containing the RGDDV pharmacophore and the RGDY(Me)R pharmacophore. These two pentapeptide sequences when properly constrained in cyclic peptides are low to sub-nanomolar inhibitors of integrin alpha(v)beta3. The peptides containing the RGDDY(Me)R sequence bind potently to integrin alphaIIb3 as well. The conformations found in H2O and in DMSO-d6 solutions are valuable for the design of peptidomimetics of these two pharmacophores. The structure-activity relationships of the RGDDV and RGDY(Me)R pharmacophores within cyclic peptides are discussed. Specifically, the orientation of surface-accessible chemical features on the ligand, such as hydrophobic, positive and negative ionizable groups, which are considered to be responsible for the desired biological activity, is focused on.     

Conformational Studies of Tachykinin Peptides Using NMR Spectroscopy

Summary A conformational analysis in water and DMSO of two tachykinin family peptides (scyliorhinin I (ScyI) and scyliorhinin II (ScyII)) was carried out by 1D and 2D NMR (DQF-COSY, TOCSY, HMQC, HMBC, NOESY and ROESY) and molecular dynamics calculation methods. In DMSO, two groups of conformations (major and minor) were obtained for both peptides based on the experimental data. The conformations proposed for ScyI represent a folded structure, which shows certain similarities to the structures reported for other NK-1 and NK-2 tachykinin agonists. In water ScyII displays a flexible, extended structure, whereas in DMSO the structure is more compact and, in the fragment from the centre to the C-terminus, several β-turns may be present.     

Solution conformation of tuftsin.

Tuftsin, a natural linear tetrapeptide (Thr-Lys-Pro-Arg) of potential antitumor activity, has been studied in DMSO-d6 solution by 2D NMR spectroscopy. 1H and 13C spectra show the presence of two families of conformations characterized by a trans or cis Lys-Pro bond, respectively. The family of conformers containing the cis peptide bond is a mixture of extended structures as expected for a short linear peptide. On the contrary, the trans isomer appears to be a rigid, folded conformer, as indicated by crucial NOEs and by the exceptionally low temperature coefficient of Arg NH. Analysis of the solution data by means of energy calculations leads to a unique structure, characterized by a Lys-Pro inverse gamma-turn.     

Comparison of helix-stabilizing effects of alpha,alpha-dialkyl glycines with linear and cycloalkyl side chains

The ability of alpha, alpha-di-n-alkyl glycines with linear and cyclic alkyl side chains to stabilize helical conformations has been compared using a model heptapeptide sequence. The conformations of five synthetic heptapeptides (Boc-Val-Ala-Leu-Xxx-Val-Ala-Leu-OMe, Xxx = Ac8c, Ac7c, Aib, Dpg, and Deg, where Ac8c = 1-aminocyclooctane-1-carboxylic acid, Ac7c = 1-aminocycloheptane-1-carboxylic acid, Aib = alpha-aminoisobutyric acid, Dpg = alpha,alpha-di-n-propyl glycine, Deg = alpha,alpha-di-n-ethyl glycine) have been investigated. In crystals, helical conformations have been demonstrated by x-ray crystallography for the peptides, R-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe, (R = Boc and acetyl). Solution conformations of the five peptides have been studied by 1H-nmr. In the apolar solvent CDCl3, all five peptides favor helical conformations in which the NH groups of residues 3-7 are shielded from the solvent. Successive NiH<-->Ni + 1H nuclear Overhauser effects over the length of the sequence support a major population of continuous helical conformations. Solvent titration experiments in mixtures of CDCl3/DMSO provide evidence for solvent-dependent conformational transitions that are more pronounced for the Deg and Dpg peptides. Solvent-dependent chemical shift variations and temperature coefficients in DMSO suggest that the conformational distributions in the Deg/Dpg peptides are distinctly different from the Aib/Acnc peptides in a strongly solvating medium. Nuclear Overhauser effects provide additional evidence for the population of extended backbone conformations in the Dpg peptide, while a significant residual population of helical conformations is still detectable in the isomeric Ac7c peptide in DMSO.     

Combined effect of the DeltaPhe or DeltaAla residue and the p-nitroanilide group on a didehydropeptides conformation

Two series of dehydropeptides of the general formulae Boc-Gly-X-Phe-p-NA, Boc-Gly-Gly-X-Phe-p-NA, Gly-X-Gly-Phe-p-NA.TFA, and Boc-Gly-X-Gly-Phe-p-NA, with X = Delta(Z)Phe and DeltaAla, were studied with NMR in DMSO and CDCl(3)-DMSO, and with CD in MeOH, MeCN, and TFE. The NMR spectra measured in DMSO suggest that peptides with the DeltaPhe residue next to Phe are folded whereas peptides with Gly between DeltaPhe and Phe are less ordered. NMR spectra of DeltaAla-containing peptides indicate that these peptides are flexible and their conformational equilibria are populated by many different conformations. The CD spectra show that conformational properties of the peptides studied are distinctly influenced by a mutual position of the dehydroamino acid residue and the p-NA group. They indicate that all dehydropeptides with the DeltaPhe residue, Boc-Gly-DeltaAla-Phe-p-NA, and Boc-Gly-Gly-DeltaAla-Phe-p-NA adopt ordered conformations in all solvents studied, presumably of the beta-turn type. The last two peptides exhibit surprising chiroptical properties. Their spectra show exciton coupling-like couplets in the region of the p-NA group absorption. This shape of CD spectra suggests a rigid, chiral conformation with a fixed disposition of the p-NA group. The CD spectra indicate that Boc-Gly-DeltaAla-Gly-Phe-p-NA and Gly-DeltaAla-Gly-Phe-p-NA.TFA are unordered, independently of the solvent.     

Nonpolar contributions to conformational specificity in assemblies of designed short helical peptides

A series of designed short helical peptides was used to study the effect of nonpolar interactions on conformational specificity. The consensus sequence was designed to obtain short helices (17 residues) and to minimize the presence of interhelical polar interactions. Furthermore, the sequence contained a heptad repeat (abcdefg), where positions a and d were occupied by hydrophobic residues Leu, Ile, or Val, and positions e and g were occupied by Ala. The peptides were named according to the identities of the residues in the adeg positions, respectively. The peptides llaa, liaa, ilaa, iiaa, ivaa, viaa, lvaa, vlaa, and vvaa were synthesized, and their characterization revealed marked differences in specificity. An experimental methodology was developed to study the nine peptides and their pairwise mixtures. These peptides and their mixtures formed a vast array of structural states, which may be classified as follows: helical tetramers and pentamers, soluble and insoluble helical aggregates, insoluble unstructured aggregates, and soluble unstructured monomers. The peptide liaa formed stable helical pentamers, and iiaa and vlaa formed stable helical tetramers. Disulfide cross-linking experiments indicated the presence of an antiparallel helix alignment in the helical pentamers and tetramers. Rates of amide proton exchange of the tetrameric form of vlaa were 10-fold slower than the calculated exchange rate for unfolded vlaa. In other work, the control of specificity has been attributed to polar interactions, especially buried polar interactions; this work demonstrated that subtle changes in the configuration of nonpolar interactions resulted in a large variation in the extent of conformational specificity of assemblies of designed short helical peptides. Thus, nonpolar interactions can have a significant effect on the conformational specificity of oligomeric short helices.     

Nuclear Overhauser effects in linear peptides. A low-temperature 500 MHz study of Met-enkephalin

Met5-enkephalin was studied in 1 mM solutions in 2H2O at room temperature and in a cryoprotective mixture (DMSOd6/2H2O, mole fraction of DMSO 0.49) in the temperature range 265-298 K. Small positive effects were observed between the ortho and meta protons of Tyr in aqueous solution at room temperature. Intraresidue effects can be made strong and negative by increasing the viscosity of the medium with a combination of cryoprotective mixtures and low temperatures. The use of mixtures with properties very close to water is very promising for conformational studies of enkephalins and of other small linear peptides.     

Comparison of conformational properties of linear and cyclic delta selective opioid ligands DTLET (Tyr-D X Thr-Gly-Phe-Leu-Thr) and DPLPE (Tyr-c[D X Pen-Gly-Phe-Pen]) by 1H n.m.r. spectroscopy

The preferential conformations of the delta selective opioid peptides DPLPE (Tyr-c[D X Pen-Gly-Phe-Pen]) and DTLET (Tyr-D X Thr-Gly-Phe-Leu-Thr) were studied by 400 MHz 1H n.m.r. spectroscopy in DMSO-d6 solution. In neutral conditions, the weak NH temperature coefficients of the C-terminal residue (Pen5 or Thr6), associated with interproton NH-NH and alpha-NH NOE's (ROESY experiments), indicated large analogies between the backbone folding tendency of both the linear and cyclic peptides. Various gamma and/or beta turns may account for these experimental data. A similar orientation of the N-terminal tyrosine related to the folded backbones is observed for the two agonists, with a probable gamma turn around the amino acid in position 2. Finally, a short distance, about 10 A, between Tyr and Phe side chains and identical structural roles for threonyl and penicillamino residues are proposed for both peptides. These results suggest the occurrence of similar conformers in solution for the constrained peptide DPLPE and the flexible hexapeptide DTLET. Therefore, it may be hypothesized that the enhanced delta selectivity of DPLPE is related to a very large conformational expense of energy needed to interact with the mu opioid receptor, a feature not encountered in the case of DTLET. These findings might allow peptides to be designed retaining a high affinity for delta opioid receptors associated with a very low cross-reactivity with mu binding sites.     

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.     

Conformational analysis of bacitracin A, a naturally occurring lariat

The proton nmr spectra of bacitracin A in H2O and DMSO-d6 have been assigned and the conformational behavior of the peptide in the two solvents has been compared. Although bacitracin A shows a conformational equilibrium between at least two conformations differing in the relative position of the cyclic and linear domains of the molecule, the spectra in water can be interpreted in terms of a preferred conformation in which the linear part is folded over the cyclic moiety and a turn is present around Ile(8)-DPhe(9).     

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.     

Conformational Studies of Tachykinin Peptides Using NMR Spectroscopy

A conformational analysis in water and DMSO of two tachykinin family peptides (scyliorhinin I (ScyI) and scyliorhinin II (ScyII)) was carried out by 1D and 2D NMR (DQF-COSY, TOCSY, HMQC, HMBC, NOESY and ROESY) and molecular dynamics calculation methods. In DMSO, two groups of conformations (major and minor) were obtained for both peptides based on the experimental data. The conformations proposed for ScyI represent a folded structure, which shows certain similarities to the structures reported for other NK-1 and NK-2 tachykinin agonists. In water ScyII displays a flexible, extended structure, whereas in DMSO the structure is more compact and, in the fragment from the centre to the C-terminus, several -turns may be present.     

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.