Kinetics of folding of guanidine-denatured hen egg white lysozyme and carboxymethyl(Cys6,Cys127)-lysozyme: a stopped-flow absorbance and fluorescence study.
The folding kinetics of hen egg white lysozyme and of a three-disulfide derivative of lysozyme [carboxymethyl(Cys6,Cys127)-hen egg white lysozyme] have been studied by absorbance- and fluorescence-detected stopped-flow techniques. A "very-fast" phase with a time constant in the millisecond range has been observed by both absorbance and fluorescence when unfolded lysozyme in 4 M guanidine hydrochloride, 100 mM phosphate buffer, and pH 2.0 is refolded at 0.5 M guanidine hydrochloride, 100 mM phosphate, and pH 6.7. Data obtained from fluorescence-detected refolding studies show that a transient intermediate is formed during the very-fast refolding phase. This intermediate is characterized by substantial quenching of tryptophan fluorescence. In addition, analysis of the fluorescence data indicates the presence of an additional "burst" phase that occurs within the dead time of the instrument, < 3 ms. The very-fast phase is not observed during the refolding of the three-disulfide derivative. In addition, the three-disulfide derivative re-attains the final native folded conformation more rapidly than the unmodified protein over the range of temperatures studied (10-20 degrees C). We conclude that, not only does the presence of the disulfide bond between Cys6 and Cys127 slow down the overall folding process of lysozyme, but it also directs the folding of lysozyme through a pathway characterized by a non-native tertiary interaction(s).
Denton
M E
ME
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Rothwarf
D M
DM
Scheraga
H A
HA
D000818:Animals; D002233:Carbocysteine; D002645:Chickens; D002942:Circular Dichroism; D004220:Disulfides; D004529:Egg White; D005260:Female; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D004220:Disulfides; D006146:Guanidines; D002233:Carbocysteine; D009113:Muramidase; D019791:Guanidine