SYNTHESIS OF CHARYBDOTOXIN AND OF 2 N-TERMINAL TRUNCATED ANALOGS - STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase syn thesis using N(alpha)-t-butyloxycarbonyl and benzyl-type side-chain pr otection. While this strategy was generally useful, the S-acetamidomet hyl protecting group used for the six cysteines was not completely sta ble under HF treatment and its subsequent removal by mercury(II) treat ment 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-pressur e liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. H-1-NMR s pectroscopy was used and a complete assignment of all the protons of t he 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 an d a similar overall three-dimensional structure, in agreement with cir cular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological a ctivity on dog epithelial Ca2+-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybd otoxin was as potent as the natural toxin on both channels. For both c hannels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while dele tion 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 f olding and stability of the structure. The structure of the shortest a nalogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid resid ues.