Identification and characterization of the direct folding process of hen egg-white lysozyme.
Refolding kinetics of hen egg-white lysozyme (HEWL) have been studied by means of the stopped-flow method with guanidinium chloride as the denaturant. We show here that the three-species model U1 in equilibrium or formed from U2 in equilibrium or formed from N (U1 and U2 = unfolded; N = native) now established for pancreatic ribonuclease A is also valid for HEWL on the basis of the following lines of evidence: (1) refolding kinetics outside the transition region are biphasic; (2) dependence of the fractional amplitude for the fast phase on the ratio of the time constants of the two phases agrees with theory; (3) unfolding kinetics outside the transition region are of single phase; (4) direct evidence for the U2 leads to U1 transformation is obtained by double-jump experiments; (5) the time constant of the binding reaction of a substrate analogue, 4-methylumbelliferyl N,-N'-diacetyl-beta-chitobioside, to HEWL molecules during refolding reaction agrees with the time constant of the direct refolding phase U2 leads to N. The characteristic properties of the nucleation-controlled reaction of refolding of small globular proteins are discussed in general. The results of the discussion are used to suggest that the direct folding process is nucleation controlled from the experimental results of the temperature dependence of the refolding rate.
D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006923:Hymecromone / Q000031:analogs & derivatives; D007700:Kinetics; D009113:Muramidase; D009844:Oligosaccharides; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D013816:Thermodynamics
D005456:Fluorescent Dyes; D006146:Guanidines; D009844:Oligosaccharides; D006923:Hymecromone; C033855:4-methylumbelliferyl-N,N-diacetylchitobioside; D009113:Muramidase; D019791:Guanidine