3-STATE MODEL FOR LYSOZYME FOLDING - TRIANGULAR FOLDING MECHANISM WITH AN ENERGETICALLY TRAPPED INTERMEDIATE

We investigated the role of a partially folded intermediate that trans iently accumulates during lysozyme folding. Previous studies had shown that the partially folded intermediate is located on a slow-folding p athway and that an additional fast direct pathway from the unfolded st ate to the native state exists. Kinetic double-jump experiments showed that the two folding pathways are not caused by slow equilibration re actions in the unfolded state. Rather, kinetic partitioning occurs ver y early in lysozyme refolding, giving the molecules the chance to ente r the direct pathway or a slow-folding channel. Fitting the guanidiniu m chloride dependencies of the refolding and unfolding reactions to an alytical solutions for different folding scenarios enables us to propo se a triangular mechanism as the minimal model for lysozyme folding ex plaining all observed kinetic reactions: [GRAPHICS] All microscopic ra te constants and their guanidinium chloride dependencies could be obta ined from the experimental data. The results suggest that population o f the intermediate during refolding increases the free energy of activ ation of the folding process. This effect is due to the increased stab ility of the intermediate state compared to the unfolded state leading to an increase in the free energy of activation (Delta GO not equal) compared to folding in the absence of populated intermediate states. T he absolute energy of the transition state is identical on both pathwa ys. The results imply that pre-formed secondary structure in the foldi ng intermediate obstructs formation of the transition state of folding but does not change the nature of the rate-limiting step in the foldi ng process. (C) 1997 Academic Press Limited.