FOLDING OF THE DISULFIDE-BONDED BETA-SHEET PROTEIN TENDAMISTAT - RAPID 2-STATE FOLDING WITHOUT HYDROPHOBIC COLLAPSE

We investigated the reversible folding and unfolding reactions of the small 74 amino acid residue protein tendamistat. The secondary structu re of tendamistat contains only beta-sheets and loop regions and the p rotein contains two disulfide bonds. Fluorescence-detected refolding k inetics of tendamistat (disulfide bonds intact) comprise of a major ra pid fast reaction (tau = 10 ms in water) and two minor slow reactions. In the fast reaction 80% of the unfolded molecules are converted to n ative protein. The two slow reactions are part of a parallel slow fold ing pathway. On this pathway the rate-limiting step in the formation o f native molecules is cis to trans isomerization of at least one of th e three trans Xaa-Pro peptide bonds. This reaction is catalyzed effici ently by the enzyme peptidyl-prolyl cis-trans isomerase. Comparison of kinetic data with equilibrium unfolding transitions shows that the fa st folding pathway follows a two-state process without populated inter mediate states. Additionally, various sensitive tests did not detect a ny rapid chain collapse during tendamistat folding prior to the acquis ition of the native three-dimensional structure. These results show th at pre-formed disulfide bonds do not prevent efficient and rapid prote in folding. (C) 1997 Academic Press Limited.