Structural resolution of the folding pathway of a protein by correlation of phi-values with inter-residue contacts.

Folding of barstar, the 10 kDalton inhibitor of the ribonuclease barnase, has been suggested to follow a nucleation-condensation model [Nölting, B., Golbik, R., Neira, J. L., Soler-Gonzalez, A. S., Schreiber, G. & Fersht, A. R. (1997). Proc. Nat. Acad. Sci. U.S.A. 94, 826-830], where structure growth starts in a particular region of the molecule, the folding nucleus. Here the structure of the diffuse nucleus and its growth in three stages, 500 micros, 1 ms and 100 ms after initiation of the folding reaction, is mapped out by using phi-values which are correlate with inter-residue contact plots. Barstar folding is initiated by a significant consolidation of interactions in and around the strand1-loop1-helix1 motif in the microsecond time scale, followed by the consolidation of helix4, which is located close to the C-terminus and does not have significant residual structure in the cold-denatured state. The non-uniform structure consolidation is most pronounced in the early stages of folding. The late folding events of barstar are characterized by a propagation of structure consolidation from the N-and C-termini towards residues located in the center of the sequence.