Role of disulfide bridges in the folding, structure and biological activity of omega-conotoxin GVIA

omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the con e shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by H-1 NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges we re synthesised using orthogonal protection of cysteine residues. In the fir st analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing t he appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was dete cted for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five ina ctive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to th e disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which pre sumably accounts for their lack of activity. The NMR studies also showed th at all five analogues had substantially altered tertiary structures, althou gh the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogu es despite the non-native disulfide pairings, This work demonstrates the im portance of the disulfides in omega-conotoxin folding and shows that the Cy s-15-Cys-26 disulfide is essential for activity in GVIA, The NMR analyses a lso emphasise that backbone chemical shifts and short- and medium-range NOE s are dictated largely by local secondary structure elements and are not ne cessarily reliable monitors of the tertiary fold. (C) 1999 Elsevier Science B.V. All rights reserved.