Conformational diversity in contryphans from Conus venom: cis-trans isomerisation and aromatic/proline interactions in the 23-membered ring of a 7-residue peptide disulfide loop.

Conformational diversity or "shapeshifting" in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis↔trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

A designed three-stranded ß-sheet in an α/ß hybrid peptide.

The incorporation of ß-amino acid residues into the antiparallel ß-strand segments of a multi-stranded ß-sheet peptide is demonstrated for a 19-residue peptide, Boc-LV(ß)FV(D)PGL(ß)FVVL(D)PGLVL(ß)FVV-OMe (BBH19). Two centrally positioned (D)Pro-Gly segments facilitate formation of a stable three-stranded ß-sheet, in which ß-phenylalanine ((ß)Phe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR-derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well-defined three-stranded ß-sheet structure in solution. Cross-strand interactions between (ß)Phe3/(ß)Phe17 and (ß)Phe3/Val15 residues define orientations of these side-chains. The observation of close contact distances between the side-chains on the N- and C-terminal strands of the three-stranded ß-sheet provides strong support for the designed structure. Evidence is presented for multiple side-chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three-stranded ß-sheet structures, which in turn influences the conformational interconversion between type I' and type II' ß-turns at the two (D)Pro-Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc-LV(ß)FV(D)PGL(ß)FVV-OMe (BBH10), which has been previously characterized as a type I' ß-turn nucleated hairpin, is shown to favour a type II' ß-turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.

NMR analysis of cross strand aromatic interactions in an 8 residue hairpin and a 14 residue three stranded ß-sheet peptide.

Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel ß-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-LFV(D)P(L)PLFV-OMe (peptide 1) favors the ß-hairpin conformation nucleated by the type II' ß-turn formed by the (D)Pro-(L)Pro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned (D)Pro-(L)Pro segments facilitate the three stranded ß-sheet formation in the 14 residue peptide Boc-LFV(D)P(L)PLFVA(D)P(L)PLFV-OMe (peptide 2) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides 1 and 2 are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the C(α)-C(ß)(χ(1)) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents.