RATE OF INTRACHAIN DIFFUSION OF UNFOLDED CYTOCHROME-C

How fast can a protein fold? The rate of initial collapse from the unf olded state to a compact structure provides one upper limit to the fol ding rate. Although hydrophobic collapse of heteropolymers is not well understood, its rate may be controlled by the rate at which contacts form between distant parts of an unfolded polypeptide chain. The rate of this intrachain diffusion has not been measured directly. However, information about that time scale is contained in the experimental res ults of Jones et al. (Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11860), who triggered the folding of reduced cytochrome c by nanosecond photol ysis of the carbon monoxide complex. Jones et al. found that the methi onine residues at positions 65 and 80 bind to the heme at position 18 at a rate of (40 mu s)(-1), while the histidine residues at positions 33 and 26 bind at a rate of (400 mu s)(-1). To identify the separate c ontributions of chain dynamics and chemical bond formation (i.e, the g eminate binding rates) to the observed rates, we have used nanosecond- resolved absorption spectroscopy to determine the bimolecular and gemi nate rates for free methionine and histidine binding to the hemepeptid e of cytochrome c under the solvent conditions of Jones et al. The rat e of his33 (and his26) binding to the heme of the intact polypeptide a ppears to be limited by a slow geminate rate and by the equilibrium pr obability that the required loop will form. spontaneously. In contrast , the binding of met65 and met80 is rate limited only by the diffusion of the polypeptide chain to form an encounter complex. The (40 mu s)( -1) rate observed by Jones et al. therefore allows us to calculate the met80-his18 intrachain diffusion rate k(D+) approximate to (35-40 mu s)(-1). From this result we estimate that the smallest intrachain loop s in a polypeptide will form in no less than similar to 1 mu s. This m ay set a limit of similar to 10(6) s(-1) on the rate of collapse of th e polypeptide chain under folding conditions. We also use the theory o f Szabo et al. (J. Chem. Phys. 1980, 72, 4350) to calculate the relati ve diffusion constant D of the heme and methionine residues, obtaining a value D approximate to 4 x 10(-7) cm(2)/s.