Folding of lysozyme.

Due to the detailed knowledge of the three-dimensional structure, chemistry and catalytic mechanism of hen egg white lysozyme, this enzyme has become a major model for the analysis of the folding pathway of globular proteins. Unfolding and folding of lysozyme are reversible processes. Unfolding is a highly cooperative event; under physiological conditions only the native and the unfolded states are stable. Folding of lysozyme involves both a cooperative and a parallel pathway. The complexities in the folding pathway arise from the collapsed state which is formed within a burst-phase in the first milliseconds of folding. In a second, fast folding phase, major parts of the secondary structures both in the alpha-domain and the beta-domain are formed. During the slow folding phase, formation of secondary structure is completed and native tertiary structure is formed in less than 1 second. Folding of reduced lysozyme combines both secondary and tertiary structure organization, as well as formation of four disulphide bonds. Analysis of formation of disulphide bonds showed that there exists a restricted search of structures in the formation of the native conformation and a nucleation in the folding pathway. The transition from a two-disulphide bond intermediate to a three-disulphide bond form appears to be the rate-limiting step in this pathway. Native-like catalytic properties depend on the correct generation of all four disulphide bonds. Folding of both denatured and denatured/reduced lysozyme is characterized by transient folding species possessing structural properties of the molten globule state: high content of secondary structure, no tertiary fold, and the appearance of hydrophobic structures.