The fast folding pathway in human lysozyme and its blockage by appropriate mutagenesis: a sequential stopped-flow fluorescence study.

In this work we were able to show that human lysozyme refolds along two parallel pathways: a fast path followed by 13% of the molecules that leads directly from a collapsed state to the native protein and a slow one for the remaining molecules that involves a partially unfolded intermediate state. However, in the refolding process of LYLA1, a chimera of human lysozyme which possesses the Ca2+-binding loop and helix C of bovine alpha-lactalbumin, the direct pathway is no longer accessible. This indicates that these structural elements, which are located in the interface region between the alpha- and beta-domain of the protein, and their interaction with the environment play an important role in the fast folding of the molecules. These results also shed some light on the conservation of folding patterns amongst structurally homologous proteins. In recent years it was often stated that structurally homologous proteins with high sequence identity follow the same folding pattern. Human lysozyme and LYLA1 have a sequence identity of 87%. However, we have shown that their folding patterns are different. Therefore, a high degree of sequence identity for two proteins belonging to the same family is not a guarantee for an identical folding pattern.