THE FOLDING PATHWAY OF A PROTEIN AT HIGH-RESOLUTION FROM MICROSECONDSTO SECONDS

We have documented the folding pathway of the 10-kDa protein barstar f rom the first few microseconds at the resolution of individual residue s from its well characterized denatured state. The denatured state had been shown from NMR to have flickering native-like structure in the f irst two of its four alpha-helices. Phi-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 e xtent. A native-like structure then is formed in a few hundred millise conds as the whole structure consolidates. Peptide fragments correspon ding to sequences containing the first two helices separately and toge ther as a helix-loop-helix motif have little helical structure under c onditions that favor folding. The early stages of folding fit the nucl eation-condensation model that aas proposed for the smaller chymotryps in inhibitor 2, which is a single module of structure and folds by two -state kinetics. The early stages of the multistate folding of the lar ger, multimodular, barnase have proved experimentally inaccessible. Th e folding pathway of barstar links those of CI2 and barnase to give a unified scheme for folding.