Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7 -37 sequences, were obtained by stepwise solid-phase synthesis using W-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(I1) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.096. Each protein was characterised chemically. structurally and functionally. 'H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall threedimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca'+-activated K' channels and on rat brain synaptosomal voltage-dependent K' channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alp fold can be engineered with only 31 amino acid residues.Protein toxins are used by venomous animals to subdue prey. These molecules, which generally have a molecular mass ranging between 2-12 kDa, are active towards different molecular targets [1-41 and are characterised by the presence of several disulfide bridges. Scorpion venoms contain a variety of proteins known to inhibit Na' and K' channels. Charybdotoxin, present as a minor component (= 0.2%) in the venom of the Israeli Lpiurus quinquestriatus scorpion, was the first discovered high-affinity inhibitor of high-conductance Ca'+-activated K' channels present in skelctal muscles [5, 61. proteins 11, 3, 4, 20, 211 with subtly different specificities and which therefore represent valuable tools in order to study diffcrcnt K' channels.We have recently reported the three-dimensional solution structure of charybdotoxin solved by two-dimensional NMR [20, 211 and the refined and detailed description of the sidechain organisation [22]. This analysis revealed the presence of a well ordered structure containing three antiparallel strands fo...