Protein folding is a complex problem. It is one of the most difficult, but exciting challenge. During our studies we isolated a new class of conotoxins which has four cysteine residues in the same location as α-conotoxins. Interestingly, the cysteine pairing is different (1-4 and 2-3) rather than that of α-conotoxins (1-3 and 2-4). These differences lead to ribbon conformation compared to globular conformation of a-conotoxins. We named this new class as λ-conotoxins. This class was discovered almost simultaneously by two other groups and was named as χ-conotoxins. We have determined the structural elements (Conformational switches) which control the disulfide pairing and folding of α- and λ/χ-conotoxins.

• We compared amino acid sequences of all known α-conotoxins and λ/χ-conotoxins. There are two important differences between these two classes: the presence of a proline residue in the first intercysteine loop in α-conotoxins, whereas λ/χ-conotoxins do not; and all α-conotoxins are amidated at their C-terminal while λ/χ-conotoxins have free carboxyl group. We identified these two play important role in disulfide pairing and folding of these conotoxins. When this proline residue is replaced by lysine or alanine residues in ImI α-conotoxin, the disulfide pairing and conformation switches to those of λ/χ-conotoxins. Similarly when the C-terminal amidation is changed to free carboxyl group, there is a switch in the conformation. These structural changes are supported by co-elution of peptides with force-folded globular and ribbon conformations as well as NMR spectral data.

• We also examined whether these conformational switches can change ribbon conformation to globular in λ/χ-conotoxins. When proline was inserted into the first intercysteine loop, indeed CMrVIa λ/χ-conotoxin switched the disulfide pairing and conformation to that of globular form. Although amidation of the C-terminal enhanced the amount of protein folded into globular conformation, by itself did not switch the conformation significantly towards globular conformation.

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