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The folding mechanism of proline-free staphylococcal nuclease (SNase (pro − )) (P11A, P31A, P42A, P47T, P56A, P117G) was investigated using the double-jump stopped-flow method (interrupted refolding). This method has enabled us to specifically monitor the amount of the native molecules during the refolding. The results indicate that the middle and slow phases observed in the refolding kinetics represent the formation of the native state (I M → N, IS → N) and that the folding mechanism of SNase (pro − ) is not represented by a single sequential pathway, but at least two parallel pathways are required for interpreting the results. For small globular proteins, the biologically active native state is formed on a millisecond to second timescale, starting from a highly disordered unfolded state. Most proteins have been found to accumu- late transient partially-folded intermediates during the refolding from the unfolded state to the native state. The existence of the partially-folded intermediates indicates that there are local minima, other than the unique global minimum, in the conformational free-energy landscape. How does a protein reach the unique global free-energy minimum (the native state) going through local free-energy minima (partially- folded intermediates) in the conformational free-energy landscape? This is an unresolved question re- garding protein folding. Staphylococcal nuclease (SNase) is a favorable system to investigate the shape of the conformational free-energy landscape of protein folding. The X-ray crystallographic structure of this small α + β protein has been reported (1), its high expression in Escherichia coli (E. coli) has been established (2,3), and this protein has been well characterized as a typical model globular protein for protein folding studies for some time (2-7). The proline-free mutant of SNase, in which all proline residues were replaced with other residues, has been constructed for the purpose of investigating the folding reaction of the protein without the intervention of slow cis/trans isomerizations of peptidyl proline bonds. Even the folding of this proline-free mutant has been found to be still a complex process, in which at least three phases of refolding (fast, middle, and slow phases) are observed (3,8). This requires at least two transient interme- diates which accumulate during the folding. Studies by the combination of pulsed hydrogen exchange and NMR have shown that the early transient intermediate contains a β-hairpin formed by strands 2 and

Parallel folding pathway of proline-free staphylococcal nuclease studied by the stopped-flow double-jump method