ULTRAFAST SIGNALS IN PROTEIN-FOLDING AND THE POLYPEPTIDE CONTRACTED STATE

To test the significance of ultrafast protein folding signals (much le ss than 1 msec), we studied cytochrome c (Cyt c) and two Cyt c fragmen ts with major C-terminal segments deleted, The fragments remain unfold ed under all conditions and so could be used to define the unfolded ba selines for protein fluorescence and circular dichroism (CD) as a func tion of denaturant concentration, When diluted from high to low denatu rant in kinetic folding experiments, the fragments readjust to their n ew baseline values in a ''burst phase'' within the mixing dead time, T he fragment burst phase reflects a contraction of the polypeptide from a more extended unfolded condition at high denaturant to a more contr acted unfolded condition in the poorer, low denaturant solvent, Hole C yt c exhibits fluorescence and CD burst phase signals that are essenti ally identical to the fragment signals over the whole range of final d enaturant concentrations, evidently reflecting the same solvent-depend ent, relatively nonspecific contraction and not the formation of a spe cific folding intermediate, The significance of fast folding signals i n Cyt c and other proteins is discussed in relation to the hypothesis of an initial rate-limiting search-nucleation-collapse step in protein folding [Sosnick, T, R,, Mayne, L, & Englander, S, W. (1996) Proteins Struct. Funct. Genet. 24, 413-426].