Analysis of Early Secodary Structure in Globular-Protein Folding.

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In order to investigate whether the presence of a transient intermediate that has folded secondary structure is a general phenomenon in globular-protein folding or not, kinetic refolding reactions of various proteins have been studied by stopped-flow circular dichroism (CD). Refolding reactions were induced by concentration jumps of guanidine hydrochloride from the unfolded to the native conditions, and resulting CD changes in peptide and side-chain regions were monitored. In all the proteins examined, i.e., lysozyme, <alpha> -lactalbumin, parvalbumin, ferricytochrome c and <beta> -lactoglobulin, there was rapid formation of secondary structure, within the dead time of the stopped-flow apparatus, before the formation of specific tertiary structure. Therefore, there is a transient intermediate formed early in the folding. In lysozyme and <alpha> -lactalbumin, their transient intermediates are similar to each other as expected from their structural homology and also essentially identical to the equilibrium unfolding intermediate of <alpha> -lactalbumin. In parvalbumin and cytochrome c, the transient intermediates have <alpha> -helical structure as expected from their structural patterns in the native state. A <beta> -structural protein, <beta> -lactoglobulin also shows a transient accumulation of the intermediate that involves <beta> -structure, comparable to the structure in the native protein, but also contains an excess of <alpha> -helix. From these results, it is concluded that the protein folding occurs in a hierarchical mechanism in which the framework of secondary structure is restored at an early stage of the reaction.