Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.     

1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.     

Conformational investigation of alpha,beta-dehydropeptides. Part. IV. Beta-turn in saturated and alpha,beta-unsaturated peptides Ac-Pro-Xaa-NHCH3: NMR and IR studies.

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Val, Phe, Leu, Abu, Ala) as well as alpha,beta-unsaturated Ac-Pro-delta Xaa-NHCH3 [delta Xaa = delta Val, (Z)-delta Phe, (Z)-delta Leu, (Z)-delta Abu] were investigated in CDCl3 and CH2Cl2 by 1H-, 13C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts delta delta for NH (Xaa) and NHCH3 on going from CDCl3 to (CD3)2SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and alpha,beta-dehydropeptides (delta Xaa) on the other. Former compounds are conformationally flexible with an inverse gamma-bend, a beta-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and alpha,beta-dehydropeptides are very similar, with the type-II beta-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The beta-turn formation propensity seems to be somewhat greater in alpha,beta-unsaturated than in heterochiral peptides, but an estimation of beta-folded conformers is risky.     

High-resolution NMR studies of fibrinogen-like peptides in solution: resonance assignments and conformational analysis of residues 1-23 of the A alpha chain of human fibrinogen

The proton resonances of the following synthetic linear human fibrinogen-like peptides were completely assigned with two-dimensional NMR techniques in solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Gly(12)-Gly(13)-Gly(14)- Val(15)-Arg(16)-Gly-Pro-Arg-Val-Val-Glu-Arg (F10), Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly(13)-Gly(14)-Val-Arg (F11), and Gly-Pro-Arg-Val-Val-Glu-Arg (F12). No predominant structure was found in the chain segment from Ala(1) to Gly(6) for F10 in both H2O and dimethyl sulfoxide. The previous suggestion that there is a hairpin loop involving residues Gly(12) to Val(15) in the A alpha chain of human fibrinogen is supported by the slow backbone NH exchange rates of Gly(14) and Val(15), by an unusually small NH chemical shift of Val(15), and by strong sequential NOE's involving this region in F10. This local chain fold within residues Asp(7) to Val(20) may place the distant Phe residue near the Arg(16)-Gly(17) peptide bond which is cleaved by thrombin.     

Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

1H NMR and CD secondary structure analysis of cell adhesion promoting peptide F-9 from laminin

A heparin binding, cell adhesion promoting domain, termed peptide F-9, from the B1 chain of human laminin, residues 641 to 660, i.e. RYVVLPRPVCFEKGMNYTVR, has been investigated by 1H NMR (500 MHz) spectroscopy and CD spectropolarimetry. While small linear peptides in water solution normally exist in a number of fluctuating conformational states, CD data analysis of peptide F9 indicates the existence of some preferred average structural populations consisting of about 30% beta-sheet, 22% beta-turn, and 6% alpha-helix. NMR structural analysis supports this observation and indicates specific sequences of preferred structural populations. Evidence for these is indicated by the presence of dNN nuclear Overhauser effect (NOE) populations and attenuated or absent d alpha N NOEs at short mixing times (0.1 s), 3J alpha N coupling constants of 5 and 10 Hz, and chemical shifts significantly removed from random coil positions. The NH2-terminal VVL sequence primarily exists in an extended chain conformation by virtue of large d alpha N NOEs and 9-10 Hz 3J alpha N coupling constants. Residues C10-N16 have turn-like or helix character with a run of dNN and d beta N NOEs and attenuated d alpha N NOEs. These midchain reversals include the lysine and asparagine residues proposed to be involved in heparin binding and N-glycosylation, respectively, to laminin peptide F-9.     

High-resolution NMR studies of fibrinogen-like peptides in solution: structural basis for the bleeding disorder caused by a single mutation of Gly(12) to Val(12) in the A alpha chain of human fibrinogen Rouen

In human fibrinogen Rouen, which is the origin of a bleedin disorder, a single amino acid is mutated from Gly(12) to Val(12) in the A alpha chain. In the previous paper of this series, this mutation was predicted to disrupt the structure of fibrinogen-like peptides bound to bovine thrombin. The structural basis of this bleeding disorder has been further assessed by studying the interaction of the following Val(12)-substituted human fibrinogen-like peptides with bovine thrombin in aqueous solution by use of two-dimensional NMR spectroscopy (including TRNOE): Ala-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala- Glu-Val(12)-Gly-Gly-Val-Arg(16)-Gly(17)-Pro-Arg-Val-NH2 (F16), Ala-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly-Val- Arg(16) (tF16), Ala-Asp-Ser-Gly-Glu-Cys(Acm)-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly-Val- Arg(16)-Gly(17)-Pro-Arg-Val-Cys(Acm)-NH2 (F17), and Ala-Asp-Ser-Gly-Glu-Cys(Acm)-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly- Val-Arg(16) (tF17). Binding of thrombin to peptides F16 and F17, and hence to tF16 and tF17 as a result of the cleavage of the Arg(16)-Gly(17) peptide bond, broadens the proton resonances of residues Asp(7) to Arg(16), suggesting that thrombin interacts specifically with this sequence of residues. Medium- and long-range TRNOE's were observed between the NH proton of Asp(7) and the C beta H protons of Ala(10) and between the ring protons of Phe(8) and the C gamma H protons of Val(12) and Val(15) in complexes of thrombin with both tF16 and tF17. A strong TRNOE, in peptides tF16 and tF17, between the C beta H protons of Glu(11) and the backbone NH proton of Val(12) was also observed. However, TRNOE's between the ring protons of Phe(8) and the C alpha H protons of Gly(14) and between the C alpha H proton of Glu(11) and the NH proton of Gly(13), previously observed in the complex of thrombin with FpA, were absent in both peptides tF16 and tF17. From incorporation of TRNOE information into distance geometry calculations, Val(12) was found to disrupt the type II beta-turn involving Glu(11) and Gly(12) that is present in complexes of thrombin with normal fibrinogen-like peptides. The positions of Gly(13) and Gly(14) in the complex are also displaced, relative to the aromatic ring of Phe(8), by the Val(12) substitution. This altered geometry presumably affects the positioning of the Arg(16)-Gly(17) bond in the active site of thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the conformation and interactions of elastin: nuclear magnetic resonance of the polyhexapeptide.

Synthesis, proton magnetic resonance and carbon-13 magnetic resonance characterizations, including complete assignments, are reported for the polyhexapeptide of elastin, HCO-Val(Ala1-Pro2-Gly3-Val4-Gly5-Val6)18-OMe. Temperature dependence of peptide NH chemical shifts and solvent dependence of peptide C-O chemical shifts have been determined in several solvents and have been interpreted in terms of four hydrogen bonded rings for each repeat of the polyhexapeptide. The more stable hydrogen bonded ring is a beta-turn involving Ala1C-O--HN-Val4. More dynamic hydrogen bonds are an 11-atom hydrogen bonded ring Gly3NH--O-C Gly5, a 7-atom hydrogen bonded ring (a gamma-turn) Gly3 C-O--NH-Gly5, and a 23-atom hydrogen bonded ring Val6inH--O-C Val6(i+1). This set of hydrogen bonds results in a right-handed beta-spiral structure with slightly more than two repeats (approximately 2.2) per turn of spiral. The beta-spiral structure is briefly discussed relative to data on the elastic fiber.     

A 1H and 13C NMR study on the role of salt-bridges in the formation of a type I beta-turn in N-acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2

The conformation of the tetrapeptide N-Acetyl-Asp7-Glu8-Lys9-Ser10-NH2, a fragment of the type I collagen alpha-1 chain N-telopeptide, has been studied by 1H and 13C NMR and circular dichroism spectroscopy. The spectroscopic evidence, based on two-dimensional, phase-sensitive NMR techniques such as COSY, ROESY, proton-carbon shift correlation and selective COLOC, indicates a strong dependence of the conformation on the experimental conditions. In CD3OH/H2O (60/40) at ca. neutral pH the tetrapeptide forms a beta-turn, stabilized by a hydrogen bond between NH(S10) and CO(D7) and a strong salt-bridge between COO-(E8) and NH3+(K9). The beta-turn is type I and appears to coexist with a non-hydrogen-bonded structure. The coexistence of these two conformers is proven by proton NMR data such as NH-NH ROEs, reduced NH-H alpha (E8) coupling constant, NH(E8) low-field shift and the temperature coefficient of NH(S10), whereas the conclusion regarding the salt-bridge is based on 13C results. In the same solvent, at a pH below the pKa of the carboxyl groups, no evidence for a conformation other than extended can be found. In aqueous solution at approximately neutral pH, evidence for the E8-K9 charge interaction is observed, but not for a hydrogen bond anywhere in the molecule.     

Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Solution conformation of a model hexapeptide containing RGD sequence.

The solution conformation of a model hexapeptide Asp-Arg-Gly-Asp-Ser-Gly (DRGDSG) containing the RGD sequence has been studied in DMSO-d6 as well as in aqueous solution (H2O:D2O/90:10%) by 1H NMR spectroscopy. The unambiguous identification of spin systems of various amino acid residues and sequence specific assignment of all proton resonances was achieved by a combination of two dimensional COSY and NOESY experiments. The temperature coefficient data of the amide proton chemical shifts in conjunction with the vicinal coupling constants, i.e. 3JNH-C alpha H, NOESY and ROESY results indicate that the peptide in both the solvents exists in a blend of conformers with beta-sheet like extended backbone structure and folded conformations. The folded conformers do not appear to be stabilised by intramolecular hydrogen bonding. Our results are consistent with the flexibility of RGD segment observed in the NMR studies on the protein echistatin containing the RGD motif (references 23-25).     

Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. I. GVGGR and GMGGQ repeats

We report solution-state pulsed field gradient nmr studies of a native sequence-derived 23-residue peptidomimetic, N alpha-acetyl-QPGVGGRQPGMGGQPGVGGRQPG-C alpha-amid, that incorporates the prevalent GVGGR and GMGGQ repeats found in the sea urchin embryo intracrystalline spicule matrix protein, SM50 (Strongylocentrotus purpuratus). These repeats are sequence homologues of elastin protein repeats (VPGVG, VGGVG, and APGVGV) and spider dragline silk protein repeats (GPGG, GQGG, and QPGYG). Using rotating frame nuclear Overhauser effect (ROE) connectivities, CH alpha proton conformational shifts, 3JNH-CH alpha coupling constants, amide temperature shift coefficients, and pulsed field gradient ROE spectroscopy solvent exchange measurements, we find that the 23-mer peptidomimetic possesses a multiple beta-turn structure in aqueous solution, in equilibria with an extended or coil structure (60% beta-turn: 40% random coil). The GVGGR sequence adopts a double beta-turn conformation that is stabilized by two hydrogen bonds (R7-->V4, R20-->V17; G6-->G3, G19-->G16). The GMGGQ region adopts a single beta-turn conformation that is stabilized by a hydrogen bond involving residues Q14 and M11. Repeating beta-turn structures, or beta-spirals, may play an important role with regard to matrix assembly, protein stability, molecular elasticity, and/or protein-crystal recognition within the spicule mineralized matrix.     

Simplification and spin-spin analysis of the side chain proton magnetic resonance spectrum of the decapeptide gramicidin S using difference scalar decoupling and biosynthesis of specifically deuterated analogs.

Biosynthesis of specifically deuterated molecules and difference scalar decoupling permitted an analysis of all C alpha-C beta spin systems of gramicidin S. Proof is presented that proton magnetic resonance spectra obtained by difference scalar decoupling yield not only spectral assignments and simplification but also accurate chemicals shifts and scalar coupling constants. The variations in (3J alpha beta) and in proton chemical shifts at temperatures over the range of -54 degrees -+66 degrees C are consistent with the internal rotation around the C alpha-C beta bonds of Val1, Orn2, Leu3, and Phe4 residues discovered using carbon 13 spectroscopy. The value (3J alpha beta) = 1.5 Hz for the proline residue is consistent with there being only one C alpha-C beta conformer. This is supported by the small temperature dependence of (3J alpha beta). However, it cannot be rigorously excluded that oscillation between a major and a minor C alpha-C beta conformation occurs for proline.     

Synthetic and conformational studies on dehydroalanine-containing model peptides

Three model dipeptides containing a dehydroalanine residue (delta Ala) at the C-terminal, Boc-X-delta Ala-NHCH3 [X = Ala, Val, and Phe,] have been synthesized and their solution conformations investigated by 1H-NMR, IR, and CD spectroscopy. NMR studies on these peptides in CDCl3 clearly indicate that the NH group of dehydroalanine is involved in an intramolecular hydrogen bond. This conclusion is supported by IR studies also. Nuclear Overhauser effect (NOE) studies are also accommodative of an inverse gamma-turn-type of conformation that is characterized by conformational angles of phi approximately -70 degrees and psi approximately +70 degrees around the X residue, and a C alpha i + 1 H-Ni + 2H interproton distance of 2.5 A. It appears that unlike dehydrophenylalanine or dehydroleucine, which tend to stabilize beta-turn type of structures occupying the i + 2 position of the turn, dehydroalanine favors the formation of an inverse gamma-turn, centered at the preceding L-residue in such solvents as CDCl3 and (CD3)2SO. A comparison of solution conformation of Boc Val-delta Ala-NHCH3 with the corresponding saturated analogue, Boc-Val-Ala-NHCH3, is also presented and shows that dehydroalanine is responsible for inducing the turn structure. It may be possible to design peptides with different preferred conformations using the suitable dehydroamino acid.