Multiple pathways on a protein-folding energy landscape: Kinetic evidence

The funnel landscape model predicts that protein folding proceeds through m ultiple kinetic pathways. Experimental evidence is presented for more than one such pathway in the folding dynamics of a globular protein, cytochrome c, After photodissociation of CO from the partially denatured ferrous prote in, fast time-resolved CD spectroscopy shows a submillisecond folding proce ss that is complete in approximate to 10(-6) s, concomitant with heme bindi ng of a methionine residue. Kinetic modeling of time-resolved magnetic circ ular dichroism data further provides strong evidence that a 50-mu s heme-hi stidine binding process proceeds in parallel with the faster pathway, imply ing that Met and His binding occur in different conformational ensembles of the protein, i.e., along respective ultrafast (microseconds) and fast (mil liseconds) folding pathways. This kinetic heterogeneity appears to be intri nsic to the diffusional nature of early folding dynamics on the energy land scape, as opposed to the tate-time heterogeneity associated with nonnative heme Ligation and proline isomers in cytochrome c.