Conformational analysis of peptide T and of its C-pentapeptide fragment

The synthetic peptide of sequence H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, termed peptide T, a competitor of the Human Immunodeficiency Virus in the binding to human T cells, and its C-terminal pentapeptide fragment, were studied by 1H-nmr in DMSO solution to determine conformational preferences. The observation of nuclear Overhauser enhancements (NOEs) for both peptides, and unusual finding for small linear peptides, allowed complete sequence-specific resonance assignments. Long-range NOEs, ring-current shifts, and the very small temperature coefficient of the Thr8 NH chemical shift suggest, for the zwitterionic form of peptide T, the presence in solution of a beta-turn involving Thr5, Asn6, Tyr7 and Thr8. This conformational feature is consistent with previous structure-activity relationship studies indicating the invariance of the same residues in several potent pentapeptide analogues. The studied pentapeptide fragment, although less structured, shows some tendency to fold even in a polar solvent such as DMSO. Preliminary chemotaxis data on some pentapeptide analogues are consistent with our structural model.     

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.     

Amino acid substitutions in the epsilon-subunit of the F1F0-ATPase of Escherichia coli

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature epsilon-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F0. Purified F1-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F1-ATPase. Partial revertant strains were isolated in which Pro-47 of the epsilon-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II beta-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III beta-turn. Space-filling models of the beta-turn (residues 46-49) in the normal, mutant and partial revertant epsilon-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the epsilon-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F1-ATPase.     

Conformational preferences of the sequential fragments of the hinge region of the human IgA1 immunoglobulin molecule

The mean solution conformation of tetrapeptide fragments spanning the hinge region of human IgA1 was investigated by CD and 13C-NMR methods. Distinct conformational differences for the partial sequences of IgA1 were found. In a series of tetrapeptides having the Thr-Pro-Pro-Thr sequence, the Pro-Pro fragment was ordered to the structure of a type II polyproline helix, but with unordered forms prevailing in the equilibria. In the case of the Pro-Pro-Thr-Pro sequence, a distinct preference for the beta-turn conformation was found. Acetylation of this tetrapeptide shifts the equilibrium towards unordered forms containing some elements of the type II polyproline helix. The peptide Thr-Pro-Ser-Pro exists predominantly in the beta-turn conformation whereas Pro-Ser-Pro-Ser-NH2 has, for the most part an unordered conformation.     

Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8

We report the conformational analysis by 1H nmr in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of analogs of the cyclic octapeptide D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol (sandostatin, octreotide). The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) contain stereochemical changes in the Thr residues in positions 6 and 8, which allow us to investigate the influence of the stereochemistry within these residues on conformation and binding affinity. The molecular dynamics simulations provide insight into the conformational flexibility of these analogs. The compounds with (S)-configuration at the C(alpha) of residue 6 adopt beta-sheet structures containing a type II' beta-turn with D-Trp in the i+1 position, and these conformations are "folded" about residues 6 and 3. The structures are very similar to those observed for sandostatin, and the disulfide bridge results in a close proximity of the H(alpha) protons of residues 7 and 2, which confirms earlier observations that a disulfide bridge is a good mimic for a cis peptide bond. The compounds with (R)-configuration at the C(alpha) of residue 6 adopt considerably different backbone conformations. The structures observed for these analogs contain either a beta-turn about residue Lys and Xaa6 or a gamma-turn about the Xaa6 residue. These compounds do not exhibit significant binding to the somatostatin receptors, while the compounds with (S) configuration in position 6 bind potently to the sst2, 3, and 5 receptors. The nmr spectra of analogs with (R) or (S) configuration at the C(alpha) of residue 8 are strikingly similar to each other. We have demonstrated that the chemical shifts of protons of residues 3, 4, 5, and 6, which are part of the type II' beta-turn, and especially the effect on the Lys gamma-protons are considerably different in active molecules as compared to inactive analogs. Since the presence of a type II' beta-turn is crucial for the binding to the receptors, the chemical shifts, the amide temperature coefficients of the Thr residue and the medium strength NOE between LysNH and ThrNH can be extremely useful as an initial screening tool to separate the active molecules from inactive analogs.     

Conformational requirement for lysine hydroxylation in collagen. Structural studies on synthetic peptide substrates of lysyl hydroxylase.

An attempt has been made to understand the conformational determinants that govern the hydroxylation of selected lysyl residues in the nascent collagen molecule by lysyl hydroxylase (EC 1.14.11.4). A series of peptide substrates of the enzyme, ranging in length from 3 to 12 residues, were synthesized. These included: tert-butyloxylcarbonyl (t-Boc)-Ile-Lys-Gly; Boc-Ala-Lys-Gly; N-acetyl-Ala-Lys-Gly-Ser; Hyp-Gly-Pro-Lys-Gly-Glu; Leu-Hyp-Gly-Ala-Lys-Gly-Glu; Gly-Phe-Hyp-Gly-Leu-Hyp-Gly-Ala-Lys-Gly-Glu; (Hyp-Gly-Pro-Lys-Gly-Glu)2; and Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly. The conformational features of these peptides were studied by spectroscopic methods so as to relate this information with the kinetic parameters for the interaction of these peptides with purified lysyl hydroxylase. Spectroscopic data, supported by conformational energy calculations, indicated that the tripeptides t-Boc-Ile-Lys-Gly and t-Boc-Ala-Lys-Gly adopt a gamma-turn structure in water and trifluoroethanol with Lys in the second position of the turn. In the tetra- and larger peptides two structures, the beta-turn and a polyproline-II (PP-II) type extended conformation, were identified. The proportions of these two structures in a given peptide depended on the polarity of the solvent. All of the peptides were hydroxylated by lysyl hydroxylase isolated from chicken embryos. In contrast, a control peptide, t-Boc-Ala-Gly-Lys which adopted a beta-turn with Lys at the end of the turn, was not hydroxylated. Competitive inhibition of the hydroxylation of protocollagen by some of the peptides showed a common binding site for these substrates in the enzyme's active site. Kinetic data on the peptides indicated improved hydroxylation rate (higher Vmax) in peptides having relatively higher beta-turn content and improved binding (lower Km) in peptides with higher content of the PP-II structure. The efficacy of the substrate was also governed by its chain length. These data suggest that the conformational criterion for lysine hydroxylation in collagen-related peptides is the presence of a "bent" structure, such as the gamma- or beta-turn at the catalytic site of lysyl hydroxylase and an "extended" PP-II type structure at the binding site(s) of the enzyme's active site. This suggestion also provides a conformational rationale for earlier observations on the substrate specificity of lysyl hydroxylase.     

Conformational preferences of sequential fragments of the hinge region of human IgA1 immunoglobulin molecule: II

The mean solution conformation of tetrapeptide fragments of the hinge region of human IgA1 molecule was investigated by CD and 13C-NMR methods. Distinct conformational differences for the partial sequences were found. Tetrapeptides with the Thr-Pro-Ser-Pro sequence were found to show a clear preference for the beta-turn conformation. Conformational equilibria of these peptides are only slightly affected by acetylation or pH changes. In the case of Pro-Thr-Pro-Ser tetrapeptides conformational equilibria are dominated by unordered forms.     

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.     

Conformational features of a synthetic model of the first extracellular loop of the angiotensin II AT1A receptor.

The angiotensin II AT1A receptor belongs to the G-protein coupled receptors (GPCRs). Like other membrane proteins, GPCRs are not easily amenable to direct structure determination by the currently available methods. The peptide encompassing the putative first extracellular loop of AT1A (residues Thr88-Leu100, el1) has been synthesized along with a cyclic model where the linear peptide has been covalently linked to a template designed to keep the distance between the peptide termini as expected in the receptor. The conformational features of the two molecules have been studied using circular dichroism and NMR techniques. The region W94PFG97 forms a type-II beta-turn and undergoes a Trp-Pro peptide bond cis-trans isomerization in both peptides confirming that these characteristics are intrinsic to el1. In addition, the presence of the spacer seems to modulate the flexibility of the peptide.     

Configurationally driven folding of model tetrapeptides containing L- or D-morpholine-3-carboxylic acids as beta-turn nucleators

The conformational analysis by NMR, IR, and molecular modeling of tetrapeptides containing morpholine-3-carboxylic acid (Mor) as a proline surrogate is presented. The relationship between the chirality of the cyclic amino acid at position i+1 and the turn propensity is maintained with respect to the reference proline-containing peptides, although marked differences in the type of folded structures were observed. The conformational profile of morpholine-containing turn peptides as a function of the chirality of the cyclic amino acid indicated that the heterochiral tetrapeptide containing the D-isomer of the cyclic amino acid is more prone to nucleate compact folded structures, although with no resemblance to the beta-turn structures of D-proline-containing peptides. Also, the solvation system proved to influence the organization of folded structures, as in the more interactive CD(3)CN the model peptides showed more compact conformations. The L-Mor-containing peptide displayed two rotamers at the Val-Mor amide bond. The trans isomer did not experience any turn structures, nor any intramolecular hydrogen-bonds, whereas the cis isomer showed a strong preference for a type VI beta-turn structure, thus providing a different conformational asset with respect to the beta-turn structure as reported for the reference L-proline model peptide.     

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.     

Conformational studies of two bradykinin antagonists by using two-dimensional NMR techniques and molecular dynamics simulations

The solution conformations of two potent antagonists of bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), [Aca(-1),DArg0,Hyp3,Thi5,DPhe7,(N-Bzl)Gly8]BK (1) and [Aaa(-1),DArg0,Hyp3,Thi5,(2-DNal)7,Thi8]BK (2), were studied by using 2D NMR spectroscopy in DMSO-d6 and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe7-(N-Bzl)Gly8 peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a beta-turn structure for the Hyp3-Gly4 residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe7 and (N-Bzl)Gly8 in analogue 1 suggests type VI beta-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb beta-turn comprising residues Ser6-Arg9 and the betaI or betaII-turn involving the Pro2-Thi5 fragment, whereas peptide 2 shows the tendency towards the formation of type I beta-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9. Moreover, the side chain of DArg0 is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.     

Conformationally constrained analogs of protein kinase inhibitor (6-22)amide: effect of turn structures in the center of the peptide on inhibition of cAMP-dependent protein kinase.

The high-affinity interaction between protein kinase inhibitor (PKI)(6-22)amide(Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly- Arg-Arg-Asn- Ala-Ile22-NH2) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr6 to Ile11 sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg15 to Ile22. Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J 264:371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a beta-turn structure might be located in the -Ala12-Ser-Gly-Arg15-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26:7641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Des12-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala12-Ser-Gly14-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The D-alanine14 PKI analog was as potent as the parent peptide, whereas the beta-alanine14 and the sarcosine14 analogs were only 10-fold less active. Several peptides that promoted a beta-turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a beta-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal beta-turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These results are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 253:414-420).     

Beta-turn formation by a six-residue linear peptide in solution.

A model peptide AAGDYY-NH2 (B1), which is found to adopt a beta-turn conformation in the TEM-1 beta-lactamase inhibitor protein (BLIP) in the TEM-1/BLIP co-crystal, was synthesized to elucidate the mechanism of its beta-turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT-IR and 1H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT-IR spectroscopy confirmed the formation of a beta-turn in solution by the model peptide. The dihedral angles [(phi3, phi3) (phi4, phi4)] of [(-52 degrees, -32 degrees ) (-38 degrees, -44 degrees )] of Gly-Asp fragment in the model peptide are consistent with those of a type III beta-turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of beta-turn formation and stability.     

The structural basis of a conserved P2 threonine in canonical serine proteinase inhibitors

Bowman-Birk inhibitors (BBIs) are a well-studied family of canonical inhibitor proteins of serine proteinases. In nature, the active region of BBIs possesses a highly conserved Thr at the P2 position. The importance of this residue has been reemphasized by synthetic BBI reactive site loop proteinomimetics. In particular, this residue was exclusively identified for active chymotrypsin inhibitors selected from a BBI template-assisted combinatorial peptide library. A further kinetic analysis of 26 P2 variant peptides revealed that Thr provides both optimal binding affinity and optimal resistance against enzymatic turnover by chymotrypsin. Herein, we report the (1)H-NMR spectroscopic study of a 5-membered sub-set of these reactive site loop peptides representing a stepwise elimination of the Thr side-chain functionalities and inversion of its side-chain chirality. The P2 Thr variant adopts a three-dimensional structure that closely mimics the one of the corresponding region of the complete protein. This validates the use of this template for the investigation of structure-function relationships. While the overall backbone geometry is similar in all studied variants, conformational changes induced by the modification of the P2 side chain have now been identified and provide a rational explanation of the kinetically observed functional differences. Eliminating the gamma-methyl group has little structural effect, whereas the elimination of the gamma-oxygen atom or the inversion of the side-chain chirality results in characteristic changes to the intramolecular hydrogen bond network. We conclude that the transannular hydrogen bond between the P2 Thr side-chain hydroxyl and the P5' backbone amide is an important conformational constraint and directs the hydrophobic contact of the P2 Thr side chain with the enzyme surface in a functionally optimal geometry, both in the proteinomimetic and the native protein. In at least four canonical inhibitor protein families similar structural arrangements for a conserved P2 Thr have been observed, which suggests an analogous functional role. Substitutions at P2 of the proteinomimetic also affect the conformational balance between cis and trans isomers at a distant Pro-Pro motif (P3'-P4'). Presented with a mixture of cis/trans isomers chymotrypsin appears to interact preferably with the conformer that retains the cis-P3' Pro-trans-P4' Pro geometry found in the parent BBI protein.     

Crystal structure of an Fab fragment in complex with a meningococcal serosubtype antigen and a protein G domain.

Many pathogens present highly variable surface proteins to their host as a means of evading immune responses. The structure of a peptide antigen corresponding to the subtype P1.7 variant of the porin PorA from the human pathogen Neisseria meningitidis was determined by solution of the X-ray crystal structure of the ternary complex of the peptide (ANGGASGQVK) in complex with a Fab fragment and a domain from streptococcal protein G to 1.95 A resolution. The peptide adopted a beta-hairpin structure with a type I beta-turn between residues Gly4P and Gly7P, the conformation of the peptide being further stabilised by a pair of hydrogen bonds from the side-chain of Asn2P to main-chain atoms in Val9P. The antigen binding site within the Fab formed a distinct crevice lined by a high proportion of apolar amino acids. Recognition was supplemented by hydrogen bonds from heavy chain residues Thr50H, Asp95H, Leu97H and Tyr100H to main-chain and side-chain atoms in the peptide. Complementarity-determining region (CDR) 3 of the heavy chain was responsible for approximately 50 % of the buried surface area formed by peptide-Fab binding, with the remainder made up from CDRs 1 and 3 of the light chain and CDRs 1 and 2 of the heavy chain. Knowledge of the structures of variable surface antigens such as PorA is an essential prerequisite to a molecular understanding of antigenic variation and its implications for vaccine design.     

Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.     

The conformational equilibria of a renin inhibitor peptide in solution

The conformational equilibrium of a decapeptide renin inhibitor (Renin Inhibitory Peptide (RIP), NH-P-H-P-F-H-F-F-V-Y-K-CO2H) in water, methanol and trifluoroethanol has been investigated. The value of a combined spectroscopic approach was apparent, with the need to define conformational states that were mixtures of conformational forms. Similarities between this study and that of the Melanin Concentrating Hormone (MCH) core peptide (5-14) are notable [1]. In water, two beta-turn conformations and an extended form were found to be in equilibrium, with cis/trans isomerism at Pro-3. Extended conformations associated with the P(II) helix and irregular forms were more favoured in aqueous environments. In MeOH and TFE, two beta-turn conformations associated with overlapping sequences and cis/trans isomerism at Pro-3 amide bond were seen to be in equilibrium. 2D ROESY and chemical-exchange cross-peaks were detected by 1H NMR and used to build up detailed models of the interconverting beta-turn conformations of RIP.