Conformational study on glycosylated asparagine-oligopeptides by NMR spectroscopy and molecular dynamics calculations.

The conformational properties of the homo oligomers of increasing chain length Boc-(Asn)(n)-NHMe (n = 2, 4, 5), (GlcNAc-beta-Asn)(n)-NHMe (n = 2, 4, 5, 8) and Boc-[GlcNAc(Ac)(3)-beta-Asn](n)-NHMe (n = 2, 4, 5) were studied by using NOE experiments and molecular dynamic calculations (MD). Sequential NOEs and medium range NOEs, including (i,i+2) interactions, were detected by ROESY experiments and quantified. The calculated inter-proton distances are longer than those characteristic of beta-turn secondary structures. Owing to the large conformational motions expected for linear peptides, MD simulations were performed without NMR constraints, with explicit water and by applying different treatments of the electrostatic interactions. In agreement with the NOE results, the simulations showed, for all peptides, the presence of both folded and unfolded structures. The existence of significant populations of beta-turn structures can be excluded for all the examined compounds, but two families of structures were more often recognized. The first one with sinusoidal or S-shaped forms, and another family of large turns together with some more extended conformations. Only the glycosylated pentapeptide shows in vacuo a large amount of structures with helical shaped form. The results achieved in water and in DMSO are compared and discussed, together with the effect of the glycosylation.     

Synthesis, metabolic stability and chemotactic activity of peptide T and its analogues

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD4 present on T4 helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4-8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10(-11)-10(-10) M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4-8)-OH abolished the biological action. In the octapeptide series, whereas the alpha-amino butyric acid (Abu) substitution for Thr4 was well tolerated, the same "slight" structural change at Thr5 or Thr8 was very detrimental. Finally, [D-Asn6]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr4 does not interfere with biological characteristics of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, conformation and biological activity of linear and cyclic Thr6-bradykinin analogues containing N-benzylglycine in place of phenylalanine.

Three linear Thr6-bradykinin analogues in which either one or both the two phenylalanine residues in the peptide sequence have been substituted by N-benzylglycine (BzlGly) and their head-to-tail cyclic analogues were synthesized and tested on an isolated rat duodenum preparation. The linear (BzlGly5,Thr6-BK, BzlGly8,Thr6-BK and BzlGly(5,8),Thr6-BK) and the cyclic (cyclo BzlGly5,Thr6-BK, cyclo BzlGly8,Thr6-BK and cyclo BzlGly(5,8),Thr6-BK) peptoid-like analogues were characterized by amino acid analysis, optical rotation, analytical HPLC and MALDI-TOF mass spectroscopy. The conformational features of both the linear and cyclic derivatives were investigated by FT-IR and CD measurements. Preliminary molecular mechanics calculations were also performed on some synthetic peptides. Pharmacological screening using the relaxation of the isolated rat duodenum preparation showed that incorporation of N-benzylglycine at positions 5 and/or 8 in the linear Thr6-BK causes a substantial decrease in potency. Comparable incorporation in cyclo Thr6-BK, at position 8, or 5 and 8, resulted in nearly inactive analogues. However, cyclo BzlGly5,Thr6-BK showed a potency which is of the same order of magnitude as for cyclo-BK and cyclo Thr6-BK.     

Conformational and structural characteristics of peptides binding to HLA-DR molecules.

A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.     

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

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

Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy.

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.     

Conformational studies on somatostatin. II. The C-terminal hexapeptide fragment

The results of a conformational study on the C terminal hexapeptide of Somatostatin are presented. Semi-empirical energy calculations and high resolution NMR methods have been used to obtain information on the conformational properties of SRIF9-14 in [2H6]dimethylsulfoxide and 2H2O. It is concluded from the energy calculations that the peptide has an averaged conformation in which semi extended and folded structures are important. Only some of the folded conformations can explain the chemical shift differences between the amino acid residues Thr10 and Thr12 as a ring current shift by the Phe11 aromatic ring on Thr10. The nonequivalence is more pronounced in dimethyl-sulfoxide (0.23--0.15 ppm) where it decreases with increasing temperature towards the temperature independent value in 2H2O (0.03 ppm). This suggests that the folded conformations are somewhat predominant in dimethylsulfoxide solutions. In 2H2O the semi extended and folded structures are statistically equally important and the peptide is more flexible. A comparison with a study on the smaller fragments SRIF10-12 and SRIF10-13 which have similar conformational properties, demonstrates the usefulness of the fragment approach in conformational studies of peptides.     

NMR analysis of human salivary mucin (MUC7) derived O-linked model glycopeptides: comparison of structural features and carbohydrate-peptide interactions.

Two series of glycopeptides with mono- and disaccharides, [GalNAc and Galbeta (1-3)GalNAc] O-linked to serine and threonine at one, two or three contiguous sites were synthesized and characterized by 1H NMR. The conformational effects governed by O-glycosylation were studied and compared with the corresponding non-glycosylated counterparts using NMR, CD and molecular modelling. These model peptides encompassing the aa sequence, PAPPSSSAPPE (series I) and APPETTAAPPT (series II) were essentially derived from a 23-aa tandem repeat sequence of low molecular weight human salivary mucin (MUC7). NOEs, chemical shift perturbations and temperature coefficients of amide protons in aqueous and nonaqueous media suggest that carbohydrate moiety in threonine glycosylated peptides (series II) is in close proximity to the peptide backbone. An intramolecular hydrogen bonding between the amide proton of GalNAc or Galbeta (1-3)GalNAc and the carbonyl oxygen of the O-linked threonine residue is found to be the key structure stabilizing element. The carbohydrates in serine glycosylated peptides (series I), on the other hand, lack such intramolecular hydrogen bonding and assume a more apical position, thus allowing more rotational freedom around the O-glycosidic bond. The effect of O-glycosylation on peptide backbone is clearly reflected from the observed overall differences in sequential NOEs and CD band intensities among the various glycosylated and non-glycosylated analogues. Delineation of solution structure of these (glyco)peptides by NMR and CD revealed largely a poly L-proline type II and/or random coil conformation for the peptide core. Typical peptide fragments of tandem repeat sequence of mucin (MUC7) showing profound glycosylation effects and distinct differences between serine and threonine glycosylation as observed in the present investigation could serve as template for further studies to understand the multifunctional role played by mucin glycoproteins.     

A 500 MHz study of peptide T in a DMSO solution

Peptide T, an octapeptide of sequence ASTTTNYT that binds to human T cells, was studied as a zwitterion in DMSOd6 solution by means of proton NMR spectroscopy at 500 MHz. The unusual dispersion of the resonances of residues of the same type (T) makes it possible to assign all resonances to specific residues by means of several 2D techniques. The non-random nature of the conformation is substantiated by the observation of sequential nuclear Overhauser enhancements (NOEs). The low value of the temperature coefficient of the chemical shift of the NH of T8 and a diagnostic NOE between the NHs of T7 and T8 hint that a beta-turn including T5, N6, Y7 and T8 is a prominent conformational feature in solution. The ring current high field shifts of the methyl group and of the NH of T8 are consistent with an interaction with the side-chain of Y7, favoured by the beta-turn.     

Peptide T exhibits a well‐defined structure in fluorinated solvent at low temperature

The structure of Peptide T was determined by solution NMR spectroscopy, under strong structure‐inducing conditions: 40% hexafluoro‐2‐propanol aqueous solution at 5 °C. Under these conditions it was possible to detect medium‐range NOEs for the first time for this peptide. This allowed a much better‐defined structure to be determined for Peptide T in comparison with earlier NMR and computational studies. Peptide structures consistent with the experimental restraints were generated using a restrained MD simulation with a full empirical force field. Residues 4–8 of Peptide T take on a well‐defined structure with a heavy atom RMSD of 0.78 Å. The structure is stabilized by hydrogen bonding to side‐chain oxygen atoms of Thr 4 and Thr 8, as well as backbone hydrogen bonding between residues 5 and 7 that forms this region into a classic γ‐turn. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of conformational properties of proline and threonine residues

The conformational role of Thr was investigated by 13C-n.m.r. and CD methods using a following series of tetrapeptides: Thr-Ala-Ala-Ala, Ala-Thr-Ala-Ala, Ala-Ala-Thr-Ala and Ala-Ala-Ala-Thr. It was found that introduction of Thr in every position of the tetraalanine peptide chain distinctly influences conformational equilibria of the peptides. An increase of beta-turn forms in conformational equilibria is induced by ionization of the terminal carboxyl group, independent of threonine position in the peptide chain. Threonine in position 1 or 3 of the peptide chain seems to have some importance for beta-turn formation in acid solution.     

Synthesis and biological properties of beta-turned Abeta(31-35) constrained analogues

A series of constrained pentapeptide analogues of the fragment Abeta(31-35) has been prepared using solid phase synthesis protocols. The results of conformational studies and surface plasmon resonance (SPR) experiments seem to indicate that the affinity of these constrained analogues for immobilized Abeta(25-35) peptide could be related to their ability to adopt a Leu34N-Ile31O beta-turn-like folded conformation.     

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.     

Dodecylphosphocholine micelles as a membrane-like environment: new results from NMR relaxation and paramagnetic relaxation enhancement analysis

To further examine to what extent a dodecylphosphocholine (DPC) micelle mimics a phosphatidylcholine bilayer environment, we performed ¹³C, ²H, and ³¹P NMR relaxation measurements. Our data show that the dynamic behavior of DPC phosphocholine groups at low temperature (12 °C) corresponds to that of a phosphatidylcholine interface at high temperature (51 °C). In the presence of helical peptides, a PMP1 fragment, or an annexin fragment, the DPC local dynamics are not affected whereas the DPC aggregation number is increased to match an appropriate area/volume ratio for accommodating the bound peptides. We also show that quantitative measurements of paramagnetic relaxation enhancements induced by small amounts of spin-labeled phospholipids on peptide proton signals provide a meaningful insight on the location of both PMP1 and annexin fragments in DPC micelles. The paramagnetic contributions to the relaxation were extracted from intra-residue cross-peaks of NOESY spectra for both peptides. The location of each peptide in the micelles was found consistent with the corresponding relaxation data. As illustrated by the study of the PMP1 fragment, paramagnetic relaxation data also allow us to supply the missing medium-range NOEs and therefore to complete a standard conformational analysis of peptides in micelles. Received: 16 April 1998 / Revised version: 19 June 1998 / Accepted: 30 July 1998     

Synthesis and biological activity of cyclic and linear analogs of C-terminal fragments of neurotensin

Using solid-phase approach, new cyclic and linear analogues of C-terminal neurotensin (NT) fragments were synthesized and their vasodepressor and miotropic activities were assayed. The cyclic structures were fixed by a peptide bond linking the lysine epsilon-amino group with the C-terminal carboxyl. Cyclization was performed by using pentafluorophenyl esters or diphenylphosphorylazide. [Phe5]-cyclo(13----6 epsilon)NT-(5-13) was found to possess high depressor activity showing certain selectivity with respect to smooth vasal muscles. Circular dichroism spectra of aqueous solutions of linear and cyclic penta- and octapeptide analogues of neurotensin indicate that the linear pentapeptide in solution adopts a folded structure, while the neurotensin fragment NT-(6-13) has an unordered structure. Cyclization of the latter fragment leads to dramatic restriction of its conformational mobility resulting in a relatively rigid structure.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

Slow O,N-acyl shift in an actinomycin-related peptide lactone

The pentapeptide lactone Cbz-(Thr-D-Val-Pro-Sar-MeAla-) was synthesized in order to observe the behavior of the unprotected lactone resulting from its hydrogenolytic deprotection. Closely related peptide lactones have been reported as intermediates in total syntheses of actinomycin D and its analogues, despite the fact that unprotected and unprotonated O-peptides of serine and threonine are known to undergo rapid O,N-acyl shift. In the present study the peptide lactone was seen to undergo a slow O,N-acyl shift, in a matter of hours, to the known cyclic pentapeptide. This contrasted with the rapid rearrangement of a model O-peptide, O-hippuryl-L-threonine methyl ester. This slowness of an O,N-acyl shift in a cyclic system presumably results from higher energy barriers of conformational origin. It explains the suitability of unprotected peptide lactones for the syntheses of actinomycins and other peptide lactone antibiotics which have appeared in the literature.     

Depsipeptide analogues of elastin repeating sequences: synthesis

Depsipeptide analogues of peptide sequences can help in elucidating the role of specific hydrogen bonds in determining the conformation in peptides. The repeating pentapeptide and hexapeptide sequences of elastin have been suggested to contain a type II beta-turn with a 4----1 hydrogen bond. Depsipeptide analogues of the repeating sequences of elastin in which this 4----1 hydrogen bond cannot exist were synthesized. A fragment condensation approach was employed in which the depsipeptide ester bond was introduced early in the synthesis. This approach proved to be effective, although the increased lability of the depsipeptide ester bond resulted in side products and low yields in some reactions.     

The Ac-RGD-NH2 peptide as a probe of slow conformational exchange of short linear peptides in DMSO

According to general belief, the conformational information on short linear peptides in solution derived at ambient temperature from NMR spectrometry represents a population-weighted average over all members of an ensemble of rapidly interconverting conformations. Usually the search for discrete conformations is concentrated at low temperatures especially when sharp NMR resonances are detected at room temperature. Using the peptide Ac-RGD-NH(2) (Ac-Arg-Gly-Asp-NH(2), Ac: acetyl) as a model system and following a new approach, we have been able to demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d(6) (DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution conditions used before their dissolution in DMSO-d(6). The conformers are stabilized by intramolecular hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their extended structures in the NMR chemical shift time scale. The reported findings provide clear evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a valuable medium for folding studies of short 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.