Hp. Feng et J. Widom, KINETICS OF COMPACTION DURING LYSOZYME REFOLDING STUDIED BY CONTINUOUS-FLOW QUASI-ELASTIC LIGHT-SCATTERING, Biochemistry, 33(45), 1994, pp. 13382-13390
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.