Comparison of the refolding of hen lysozyme from dimethyl sulfoxide and guanidinium chloride.

The folding of hen egg white lysozyme is complex, involving parallel pathways and distinct folding domains [Radford, S.E., Dobson, C.M., & Evans, P.A. (1992) Nature 358, 302-307]. In the present work the refolding of this protein from two denatured states that have different conformational properties, one generated by the presence of guanidinium chloride (GdmCl) and the other by dimethyl sulfoxide (DMSO), has been examined. Refolding was initiated by rapid dilution and followed by hydrogen-exchange pulse labeling, stopped-flow circular dichroism (CD) in the near-ultraviolet region, and stopped-flow fluorescence experiments. When the final refolding conditions were identical (545 mM GdmCl, 8% (v/v) DMSO, and 20 mM sodium acetate, pH 5.5, 20 degrees C), the folding behavior from the different denatured states monitored by near-UV CD and hydrogen-exchange pulse labeling was indistinguishable. These experiments indicate that the folding process of hen lysozyme is not significantly dependent on the nature of the two denatured states. The complexities in the pathway, therefore, appear to arise from properties of the collapsed state which is formed within the first few milliseconds of refolding. The kinetics of folding were found to be dependent on the concentration of DMSO in the final refolding buffer, although the fundamental properties of the pathway, including the existence of parallel events and distinct folding domains, are preserved under all the conditions studied. Inclusion of DMSO in the refolding buffer increases the rate of formation of native-like structure and of the native state itself. This could result from destablization of species formed early in folding, allowing them to rearrange more rapidly to permit productive folding to proceed. The results indicate that examination of a wide range of conditions will contribute substantially to a more complete understanding of protein folding pathways.