Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyc lic polypeptides in which the major structural features are a circular pept ide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring f amily of circular proteins and have prompted us to explore the concept of a cyclic permutation, i.e. opening the backbone of a cross-linked circular pr otein in topologically permuted ways. We have synthesized the complete suit e of acyclic permutants of kalata B1 and examined the effect of acyclic per mutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and t hese involve disruption of the embedded ring in the cystine knot. Surprisin gly, it is possible to disrupt regions of the p-sheet and still allow foldi ng into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the na tive peptide being retained in all but two cases, none of the acyclic permu tants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemo lytic activity.