Folding of aspartokinase-homoserine dehydrogenase I is dominated by tertiary interactions

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Abstract
In the presence of guanidine hydrochloride concentrations > 2 M, aspartokinase-homoserine dehydrogenase I [from Escherichia coli] remains sufficiently soluble so that the fluorescence and circular dichroism of the protein can be measured. Both parameters show that, up to 3 M guanidine hydrochloride, the protein exists in a stable folded state which possesses a large amount of secondary structure and buried tryptophan residues. This intermediate species is probably monomeric; it is reversibly unfolded by guanidine hydrochloride concentrations of 3-4 M. This folded species is formed rapidly from unfolded protein when the denaturant is diluted out and this rapid folding step precedes all the reactivation steps described previously. The existence of a stable monomeric and folded intermediate indicates that the tertiary interactions have a major contribution to the stability of the native structure of aspartokinase-homoserine dehydrogenase I. Similar measurements were performed on 2 complementary nonoverlapping fragments: a kinase fragment corresponding to the N-terminal third and a dehydrogenase fragment corresponding to the C-terminal 2/3 of the polypeptide chain. Both fragments exist in a stable folded state up to 2.5 M guanidine hydrochloride. Both fragments show cooperative unfolding transitions between 2.5 and 4 M denaturant. The stability of the folded state of a given region is about the same in an isolated fragment and in the entire chain of aspartokinase-homoserine dehydrogenase I; an equimolar mixture of these 2 fragments and the intact chain would give about the same results. Folding of the kinase and dehydrogenase regions apparently occurs independently with a single subunit of the entire protein.

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