KINETICS OF LYSOZYME REFOLDING - STRUCTURAL CHARACTERIZATION OF A NONSPECIFICALLY COLLAPSED STATE USING TIME-RESOLVED X-RAY-SCATTERING

We report time-resolved small angle X-ray scattering (SAXS) studies of the structural characteristics of the collapsed state of lysozyme fro m hen-egg white (HEL) obtained on initiating refolding by rapidly chan ging solvent conditions from 8 M to 1.1 M urea at pH 2.9. At this redu ced pH the lifetime, of about one second, of the non-specifically coll apsed ensemble is considerably prolonged relative to its value at pH 5 .2. The SAXS studies are combined with time resolved measurements of t ryptophan fluorescence and of the rate of formation of native molecule s using interrupted refolding experiments. We observe large burst phas e changes in intrinsic tryptophan fluorescence and in the radius of gy ration (R-g) which is reduced from 22 Angstrom in the fully unfolded s tate to similar to 19 to 20 Angstrom. Subsequent decrease of the R-g t o the value for native lysozyme (15 Angstrom) follows the time course of formation of native molecules. Single exponential fits to the singu lar value decomposition (SVD) components of the SAXS data allow recons truction of the SAXS profile at early time points of refolding. The re sults of this analysis suggest a globular shape of the collapsed state . A similar fit to the forward scattering amplitude, I(0), suggests th at the collapsed state has a solvent accessible surface area which is considerably increased relative to that of the native protein. These r esults show directly that the non-specifically collapsed state formed during the burst phase in lysozyme refolding indeed represents a molec ular compaction and a change in shape from a fully denatured random co il state (albeit restricted by disulfide bonds) to an ensemble of glob ular conformations which, however, have not yet formed a solvent-prote cted hydrophobic core. (C) 1998 Academic Press Limited.