The origin of the α-domain intermediate in the folding of hen lysozyme (original) (raw)

1998, Journal of Molecular Biology

Stopped-¯ow¯uorescence and circular dichroism spectroscopy have been used in conjunction with quenched-¯ow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 C and 50 C. At 50 C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 C, although folding occurs signi®cantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 C, having persistent native-like structure in the a-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 C. In addition, the characteristic maximum in negative ellipticity and the minimum in¯uorescence intensity observed in far UV CD and intrinsic¯uorescence experiments at 20 C, respectively, are not seen at 50 C. Addition of 2 M NaCl to the refolding buffer at 50 C, however, regenerates both the hydrogen exchange and optical properties associated with the a-domain intermediate but has no signi®cant effect on the overall refolding kinetics. Together with previous ®ndings, these results indicate that non-native interactions within the a-domain intermediate are directly responsible for the unusual optical properties observed during refolding, and that this intermediate accumulates as a consequence of its intrinsic stability in a folding process where the formation of stable structure in the b-domain constitutes the rate-limiting step for the majority of molecules.