title: Kinetics of compaction during lysozyme refolding studied by continuous-flow quasielastic light scattering.
abstract: We recently developed an experiment, termed continuous-flow quasielastic light scattering (QLS), that is capable of monitoring the time evolution of the hydrodynamic diameter of macromolecules or macromolecular assemblies in solution. Here we report the use of this method to directly monitor the kinetics of compaction of the polypeptide chain of hen egg white lysozyme (HEWL) when protein refolding is initiated by 10-fold dilution from 5 M guanidine hydrochloride (GuHCl) at pH 1.5, 23 degrees C. Previously, such information could only be obtained indirectly, by analysis of the kinetics of binding ans release of a fluorescent probe dye. Refolding was also monitored by UV difference absorption spectroscopy to characterize the time scale of the formation of the native environment around the aromatic side chains under the same conditions used in the continuous-flow QLS experiments. We find that HEWL becomes compact within 1 s after the initiation of refolding, the shortest time that is accessible with our first-generation instrument. This time scale is shorter than that for the recovery of the native absorbances in the aromatic region. These results provide direct evidence that the intermediate on the folding pathway of lysozyme is compact. The implications of these results for models of protein folding are discussed.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
authors.forename: H P
authors.initials: HP
authors.lastname: Feng
authors.forename: J
authors.initials: J
authors.lastname: Widom
mesh_terms: D000818:Animals; D002645:Chickens; D004527:Egg Proteins; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D004527:Egg Proteins; D006146:Guanidines; D046911:Macromolecular Substances; D009113:Muramidase; D019791:Guanidine