NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Two-dimensional NMR studies of the antimicrobial peptide NP-5

Nearly complete proton resonance assignment of the rabbit antimicrobial peptide NP-5 has been made from two-dimensional NMR data taken at a single temperature. The assignment procedure involved acquisition of phase-sensitive double-quantum-filtered correlation spectra, relayed coherence-transfer spectra, total correlation (homonuclear Hartmann-Hahn) spectra, double- and triple-quantum spectra, and nuclear Overhauser effect spectra. The combination of these complementary experiments simplified and accelerated resonance assignment of the peptide. Individual assignments were made at 20 degrees C for all amide and C alpha protons in the peptide, and for all nonlabile side-chain protons on 26 of the 33 amino acid residues in NP-5. Analysis of the proton-proton nuclear Overhauser effect connectivities, the slowly exchanging amide protons, and the proton chemical shifts in NP-5 indicates that the peptide has a stable, ordered structure in solution. These data also indicate that residues 19-29 in NP-5 are involved in an antiparallel beta-sheet that has a hairpin conformation.     

Direct inactivation of viruses by human granulocyte defensins.

Human neutrophils contain a family of microbicidal peptides known as defensins. One of these defensins, human neutrophil peptide (HNP)-1, was purified, and its ability to directly inactivate several viruses was extensively tested. Herpes simplex virus (HSV) types 1 and 2, cytomegalovirus, vesicular stomatitis virus, and influenza virus A/WSN were inactivated by incubation with HNP-1. Two nonenveloped viruses, echovirus type 11 and reovirus type 3, were resistant to inactivation. Purified homologous peptides HNP-2 and HNP-3 were found to have HSV-1-neutralizing activities approximately equal to that of HNP-1. Inactivation of HSV-1 by HNP-1 depended on the time, temperature, and pH of incubation. Antiviral activity was abrogated by low temperature or prior reduction and alkylation of the defensins. Addition of serum or serum albumin to the incubation mixtures inhibited neutralization of HSV-1 by HNP-1. We used density gradient sedimentation techniques to demonstrate that HNP-1 bound to HSV-1 in a temperature-dependent manner. We speculate that binding of defensin peptides to certain viruses may impair their ability to infect cells.     

Three-dimensional structure of RK-1: a novel alpha-defensin peptide

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of RK-1, an antimicrobial peptide from rabbit kidney recently discovered from homology screening based on the distinctive physicochemical properties of the corticostatins/defensins. RK-1 consists of 32 residues, including six cysteines arranged into three disulfide bonds. It exhibits antimicrobial activity against Escherichia coli and activates Ca(2+) channels in vitro. Through its physicochemical similarity, identical cysteine spacing, and linkage to the corticostatins/defensins, it was presumed to be a member of this family. However, RK-1 lacks both a large number of arginines in the primary sequence and a high overall positive charge, which are characteristic of this family of peptides. The three-dimensional solution structure, determined by NMR, consists of a triple-stranded antiparallel beta-sheet and a series of turns and is similar to the known structures of other alpha-defensins. This has enabled the definitive classification of RK-1 as a member of this family of antimicrobial peptides. Ultracentrifuge measurements confirmed that like rabbit neutrophil defensins, RK-1 is monomeric in solution, in contrast to human neutrophil defensins, which are dimeric.     

Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family

Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.     

Antibacterial activities and conformations of synthetic alpha-defensin HNP-1 and analogs with one, two and three disulfide bridges.

Structure and biological activities of synthetic peptides corresponding to human alpha-defensin HNP-1, AC1YC2RIPAC3IAGERRYGTC4IYQGRLWAFC5C6 with the S-S connectivities: C1-C6, C2-C4, C3-C5, and its variants with one, two and three disulfide bridges were investigated. Oxidation of synthetic, reduced HNP-1 yielded a peptide with S-S connectivities C1-C3, C2-C4 and C5-C6, and not with the S-S linkages as in naturally occurring HNP-1. Selective protection of cysteine sulfhydryls was necessary for the formation of S-S bridges as in native HNP-1. Likewise, oxidation of peptide encompassing the segment from C2 to C5, resulted in the S-S linkages C2-C3 and C4-C5 instead of the expected linkage C2-C4 and C3-C5. Antibacterial activities were observed for all peptides, irrespective of how the S-S bridges were linked. Linear peptides without S-S bridges were inactive. Circular dichroism (CD) spectra suggest that peptides constrained by one and two S-S bridges do not form rigid beta-sheet structures in an aqueous environment. The spectrum of HNP-1 in an aqueous environment suggests the presence of a beta-hairpin conformation. In the presence of lipid vesicles, the S-S constrained peptides tend to adopt a beta-structure. Although the S-S connectivities observed in HNP-1 may be necessary for other physiological activities, such as chemotaxis, they are clearly not essential for antibacterial activity.     

Solution structures of the rabbit neutrophil defensin NP-5

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.     

Design and NMR conformational study of a beta-sheet peptide based on Betanova and WW domains

A good approach to test our current knowledge on formation of protein beta-sheets is de novo protein design. To obtain a three-stranded beta-sheet mini-protein, we have built a series of chimeric peptides by taking as a template a previously designed beta-sheet peptide, Betanova-LLM, and incorporating N- and/or C-terminal extensions taken from WW domains, the smallest natural beta-sheet domain that is stable in absence of disulfide bridges. Some Betanova-LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt beta-sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest beta-sheet population according to the CD spectra, named 3SBWW-2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW-2 adopts a well defined three-stranded antiparallel beta-sheet structure with a disordered C-terminal tail. To discern between the contributions to beta-sheet stability of strand residues and the C-terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW-2 but lacking the C-terminal extension, named Betanova-LYYL, was also investigated. beta-Sheet stability in these two peptides, in the parent Betanova-LLM and in WW-P, a prototype WW domain, decreased in the order WW-P > 3SBWW-2 > Betanova-LYYL > Betanova-LLM. Conclusions about the contributions to beta-sheet stability were drawn by comparing structural properties of these four peptides.     

Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.     

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional (1)H NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta-hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta-sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta-sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.     

Spectroscopic studies of structural changes in two beta-sheet-forming peptides show an ensemble of structures that unfold noncooperatively

A characterization of the conformation and stability of model peptide systems that form beta-sheets in aqueous solutions is considerably important in gaining insights into the mechanism of beta-sheet formation in proteins. We have characterized the conformation and equilibrium folding and unfolding of two 20-residue peptides whose NMR spectra suggest a three-stranded beta-sheet topology in aqueous solution: Betanova [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256] and (D)P(D)P with d-Pro-Gly segments at the turns [Schenck, H. L., and Gellman, S. H. (1998) J. Am. Chem. Soc. 120, 4869-4870]. Both circular dichroism (CD) and infrared measurements indicate only 20-26% beta-sheet-like structure at 5 degrees C for Betanova and 42-59% beta-sheet for (D)P(D)P. For both peptides, the CD and infrared spectra change nearly linearly with increasing temperatures (or urea concentrations) and lack a sigmoidal signature characteristic of cooperative unfolding. Fluorescence resonance energy transfer (FRET) measurements between donor and acceptor molecules attached to the two ends confirm that Betanova is largely unstructured even at 10 degrees C; the average end-to-end distance estimated from FRET is closer to that of a random coil than a structured beta-sheet. In (D)P(D)P, the FRET results indicate a more compact structure that remains compact even at high temperatures (approximately 80 degrees C) or high urea concentrations (approximately 8 M). These results indicate that both these peptides access an ensemble of conformations at all temperatures or denaturant concentrations, with no significant free energy barrier separating the "folded" and "unfolded" conformations.     

Chemoattraction of macrophages, T lymphocytes, and mast cells is evolutionarily conserved within the human alpha-defensin family

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated alpha- and beta-defensins. Human alpha-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1-4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although alpha-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Galphai proteins and MAPK as signal transducers. Alpha-defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to beta-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and G?6976, we observed a PKC-independent functional desensitization to occur between human alpha-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This alpha-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human beta-defensins. Conversely, alpha-defensins desensitized beta-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human alpha-defensin family and tightly regulated by beta-defensins.     

NMR studies of toxin III from the sea anemone Radianthus paumotensis and comparison of its secondary structure with related toxins

Nearly complete assignments of the proton nuclear magnetic resonance (NMR) spectrum of the polypeptide toxin III from the sea anemone Radianthus paumotensis (RP) are presented. The secondary structures of the related toxins RP II and RP III are described and are compared with each other and with another related toxin ATX Ia from Anemonia sulcata [Widmer, H., Wagner, G., Schweitz, H., Lazdunski, M., & Wüthrich, K. (1988) Eur. J. Biochem. 171, 177-192]. All of these proteins contain a highly twisted four-strand antiparallel beta-sheet core connected by loops of irregular structure. From the work done with AP-A from Anthopleura xanthogrammica [Gooley, P. R., & Norton, R. S. (1986) Biochemistry 25, 2349-2356], it is clear that this homologous toxin also has the same basic core. Some small differences are seen in the structures of these toxins, particularly in the position of the N-terminal residues that form one of the outside strands of the beta-sheet. In addition, the R. paumotensis toxins are two residues longer, extending the third strand of sheet containing the C-terminal residues. A comparison of chemical shifts for assigned residues is also presented, in general supporting the similarity of structure among these proteins.     

De novo design of a monomeric three-stranded antiparallel beta-sheet

Here we describe the NMR conformational study of a 20-residue linear peptide designed to fold into a monomeric three-stranded antiparallel beta-sheet in aqueous solution. Experimental and statistical data on amino acid beta-turn and beta-sheet propensities, cross-strand side-chain interactions, solubility criteria, and our previous experience with beta-hairpins were considered for a rational selection of the peptide sequence. Sedimentation equilibrium measurements and NMR dilution experiments provide evidence that the peptide is monomeric. Analysis of 1H and 13C-NMR parameters of the peptide, in particular NOEs and chemical shifts, and comparison with data obtained for two 12-residue peptides encompassing the N- and C-segments of the designed sequence indicates that the 20-residue peptide folds into the expected conformation. Assuming a two-state model, the exchange kinetics between the beta-sheet and the unfolded peptide molecules is in a suitable range to estimate the folding rate on the basis of the NMR linewidths of several resonances. The time constant for the coil-beta-sheet transition is of the order of several microseconds in the designed peptide. Future designs based on this peptide system are expected to contribute greatly to our knowledge of the many factors involved in beta-sheet formation and stability.     

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.     

Human Neutrophil Peptide-1 (HNP-1): A New Anti-Leishmanial Drug Candidate

The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs) have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1), which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1) against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG–treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-β concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR), we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells.     

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1,3)-Ac2-alpha-D-GalN3+ ++]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CalphaH chemical shift perturbations, 3JNH:CalphaH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.     

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).