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 21 ms because the folding of denatured proteins is usually initiated by mixing 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 suitable protein. We are able to detect early events in the folding of mutants of barstar, the polypeptide inhibitor of barnase. A preliminary characterization of the fast phase from spectroscopic and 4D-value analysis indicates that it is a transition between two relatively solventexposed states with little consolidation of structure.The pathway by which a particular protein folds will be resolved experimentally when the structures of all stable, metastable, and transition states adopted by the protein during the process have been characterized structurally and energetically. The only way to analyze the structures of transition states is by kinetics, and the details of their structure and energetics can be gleaned from the kinetics of folding of proteins whose structures have been carefully altered by protein engineering (1-6). This protein engineering procedure has been used to characterize transition states and intermediates on a time scale of .1 ms (1-6), the time resolution of the stopped-flow and other rapid mixing techniques employed so far in these and most other studies (e.g., refs. 7-9). This is too slow to allow detection of the early events that initiate folding. To study faster events, kinetic techniques must be employed that eliminate the time delays arising from rapid mixing techniques. Relaxation methods, by which a preexisting equilibrium between denatured and folded states is rapidly perturbed by a change in physical or chemical conditions, may be used to extend the time range. It is not easy, however, to find methods that can readily cause a denatured state of a protein to renature. Millisecond time resolution has been achieved by using a repetitive pressure-perturbation method (10). Laser flash photolysis has been applied to dissociate CO from CO-bound cytochrome c, with concomitant denaturation (11). However, rapid CO rebinding, binding of non-native ligands, and possibly aggregation prevented the complete transition to the native state (11). A more generally applicable method for studying fast events would be to raise rapidly the temperature of a cold-denatured protein, since cold denaturation is a common phenomenon of globular proteins under suitable experimental conditions (12-15). Temperature jump (T-jump) by electrical discharge (16-20) and laser-induced heating would thus enable microsecond and nanosecond time resolutions, respectively.The structure of barstar (21), the inhibitor of th...

We have documented the folding pathway of the 10-kDa protein barstar from the first few microseconds at the resolution of individual residues from its well characterized denatured state. The denatured state had been shown from NMR to have f lickering native-like structure in the first two of its four ␣-helices. ⌽-value analysis shows that the first helix becomes substantially consolidated as the intermediate is formed in a few hundred microseconds, as does the second to a lesser extent. A native-like structure then is formed in a few hundred milliseconds as the whole structure consolidates. Peptide fragments corresponding to sequences containing the first two helices separately and together as a helix-loop-helix motif have little helical structure under conditions that favor folding. The early stages of folding fit the nucleationcondensation model that was proposed for the smaller chymotrypsin inhibitor 2, which is a single module of structure and folds by two-state kinetics. The early stages of the multistate folding of the larger, multimodular, barnase have proved experimentally inaccessible. The folding pathway of barstar links those of CI2 and barnase to give a unified scheme for folding.

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