Fast and slow tracks in lysozyme folding: insight into the role of domains in the folding process.

The folding of lysozyme involves parallel events in which hydrogen exchange kinetics indicate the development of persistent structure at very different rates. We have monitored directly the kinetics of formation of the native molecule by the binding of a fluorescently labelled inhibitor, MeU-diNAG (4-methylumbelliferyl-N,N'-diacetyl-beta-D-chitobioside). The data show that native character monitored in this way also develops with different timescales. Although the rate determining step on the slow pathway (approximately 75% of molecules at pH 5.5, 20 degrees C) can be attributed to the need to reorganise structure formed early in the folding process, the data indicate that the rate determining step on the fast track (involving approximately 25% of molecules) involves the docking of the two constituent domains of the protein. In the fast folding track the data are consistent with a model in which each domain forms persistent structure prior to their docking in a locally cooperative manner on a timescale comparable to the folding of small single domain proteins.