SUBMILLISECOND EVENTS IN PROTEIN-FOLDING

The pathway of protein folding is now being analyzed at the resolution of individual residues by kinetic measurements on suitably engineered mutants. The kinetic methods generally employed for studying folding are typically limited to the time range of greater than or equal to 1 ms because the folding of denatured proteins is usually initiated by m ixing them with buffers that favor folding, and the dead time of rapid mixing experiments is about a millisecond. We now show that the study of protein folding may be extended to the microsecond time region by using temperature-jump measurements on the cold-unfolded state of a su itable protein. We are able to detect early events in the folding of m utants of barstar, the polypeptide inhibitor of barnase. A preliminary characterization of the fast phase from spectroscopic and Phi-value a nalysis indicates that it is a transition between two relatively solve nt-exposed states with little consolidation of structure.