A specific transition state for S-peptide combining with folded S-protein and then refolding

We measured the folding and unfolding kinetics of mutants for a simple prot ein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form r ibonuclease S analogues: initially, S-peptide is disordered whereas S-prote in is folded. The fluorescent probe provides a convenient spectroscopic pro be for the reaction. The association rate constant, k(on), and the dissocia tion rate constant, k(off), were both determined for two sets of mutants. T he dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows k(o n) and k(off) to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding a nd refolding. The first set of mutants tests the role of the alpha-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the alp ha-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log k(on) vs. log K-d for this series of mut ants is linear over a very wide range, with a slope of -0.3, indicating tha t almost all of the molecules fold via a transition state involving the hel ix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, whi ch are known to be important for binding S-peptide to S-protein and which a lso contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transiti on state. The results provide a remarkably clear description of a folding t ransition state.