COMPARISON OF THE REFOLDING OF HEN LYSOZYME FROM DIMETHYL-SULFOXIDE AND GUANIDINIUM CHLORIDE

The folding of hen egg white lysozyme is complex, involving parallel p athways and distinct folding domains [Radford, S. E., Dobson, C. M., a nd Evans, P. A. (1992) Nature 358, 302-307]. In the present work the r efolding of this protein from two denatured states that have different conformational properties, one generated by the presence of guanidini um chloride (GdmCl) and the other by dimethyl sulfoxide (DMSO), has be en 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 experi ments. When the final refolding conditions were identical (545 mM GdmC l, 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 nea r-UV CD and hydrogen-exchange pulse labeling was indistinguishable. Th ese experiments indicate that the folding process of hen lysozyme is n ot significantly dependent on the nature of the two denatured states. The complexities in the pathway, therefore, appear to arise from prope rties of the collapsed state which is formed within the first few mill iseconds of refolding. The kinetics of folding were found to be depend ent on the concentration of DMSO in the final refolding buffer, althou gh the fundamental properties of the pathway, including the existence of parallel events and distinct folding domains, are preserved under a ll the conditions studied. Inclusion of DMSO in the refolding buffer i ncreases the rate of formation of native-like structure and of the nat ive state itself. This could result from destablization of species for med early in folding, allowing them to rearrange more rapidly to permi t 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.