THE LENGTH OF A SINGLE TURN CONTROLS THE OVERALL FOLDING RATE OF 3-FINGERED SNAKE TOXINS

Snake curaremimetic toxins are shea all-beta proteins, containing seve ral disulfide bonds which largely contribute to their stability. The f our disulfides present in snake toxins make a ''disulfide beta-cross'' -fold that was suggested to be a good protein folding template. Previo us studies on the refolding of snake toxins (Menez, A. et al. (1980) B iochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggeste d that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and c lose to the ''disulfide beta-cross''. To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxin s, toxin alpha (Naja nigricollis) and erabutoxin b (Laticauda semifasc iata), and two synthetic homologues, the alpha mutants, alpha 60 and a lpha 62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one resid ue in the turn length that reproduced the natural heterogeneity at tha t locus. The refolding was studied by electrospray mass spectrometry ( ESMS) time-course analysis. This analysis permitted both the identific ation and quantitation of the population of intermediates present duri ng the process. All toxins were shown to share the same sequential sch eme for disulfide bond formation despite large differences in their re folding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed dif ferences in the refolding rate of toxins.