Kinetics of secondary structure recovery during the refolding of reduced hen egg white lysozyme.
We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 microM oxidized glutathione, and 20 microM reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi- or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.
Roux
P
P
Unité de Biochimie Cellulaire, CNRS URA 1129, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Delepierre
M
M
Goldberg
M E
ME
Chaffotte
A F
AF
D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D005260:Female; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D009113:Muramidase; D005978:Glutathione; D019791:Guanidine; D004229:Dithiothreitol; D019803:Glutathione Disulfide