Two-State Folding of Lysozyme Versus Multiple-State Folding of α-Lactalbumin Illustrated by the Technique of Disulfide Scrambling

Abstract

The folding of lysozyme and of α-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of αLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and αLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys⁶-Cys³⁰, Cys⁶⁴-Cys⁷⁶, Cys⁸⁰-Cys⁹⁴, Cys¹¹⁵-Cys¹²⁷) and αLA (Cys⁶-Cys²⁸, Cys⁶¹-Cys⁷³, Cys⁷⁷-Cys⁹¹, Cys¹¹¹-Cys¹²⁰) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of αLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of αLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of αLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.