KINETICS OF COMPACTION DURING LYSOZYME REFOLDING STUDIED BY CONTINUOUS-FLOW QUASI-ELASTIC LIGHT-SCATTERING

We recently developed an experiment, termed continuous-flow quasielast ic light scattering (QLS), that is capable of monitoring the time evol ution of the hydrodynamic diameter of macromolecules or macromolecular assemblies in solution. Here we report the use of this method to dire ctly monitor the kinetics of compaction of the polypeptide chain of he n egg white lysozyme (HEWL) when protein refolding is initiated by 10- fold dilution from 5 M guanidine hydrochloride (GuHCl) at pH 1.5, 23 d egrees C, Previously, such information could only be obtained indirect ly, by analysis of the kinetics of binding and release of a fluorescen t probe dye. Refolding was also monitored by UV difference absorption spectroscopy to characterize the time scale of the formation of the na tive environment around the aromatic side chains under the same condit ions used in the continuous-flow QLS experiments. We find that HEWL be comes compact within 1 s after the initiation of refolding, the shorte st time that is accessible with our first-generation instrument. This time scale is shorter than that for the recovery of the native absorba nces in the aromatic region. These results provide direct evidence tha t the intermediate on the folding pathway of lysozyme is compact. The implications of these results for models of protein folding are discus sed.