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/oxidize d hen lysozyme recovered its native secondary structure, while the red uced protein remained fully unfolded, To investigate the role played b y disulfide bridges in the acquisition of the secondary structure at l ater stages of the renaturation/oxidation, the complete refolding of r educed lysozyme was studied, This was done in a renaturation buffer co ntaining 0.5 M guanidinium chloride, 60 mu M oxidized glutathione, and 20 mu M 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 b y activity, compactness, stability, and several spectroscopic measurem ents, The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism si gnals, When bi- or triphasic sequential models were fitted to the expe rimental data, the first two phases had the same calculated rate const ants for all the signals showing that, within the time resolution of t hese experiments, the folding/oxidation of hen lysozyme is highly coop erative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.