Mechanism of renaturation of a large protein, aspartokinase-homoserine dehydrogenase.
The renaturation of aspartokinase-homoserine dehydrogenase and of some of its smaller fragments has been investigated after complete unfolding by 6 M guanidine hydrochloride. Fluorescence measurements show that a major folding reaction occurs rapidly (in less than a few seconds) after the protein has been transferred to native conditions and results in the shielding of the tryptophan residues from the aqueous solvent; this step also takes place in the fragments and probably corresponds to the independent folding of different segments along the polypeptide chain. The reappearance of the kinase activity, which is an index of the formation of "native" structure within a single chain, is much slower (a few minutes) and has the following properties: it is involved in a kinetic competition with the formation of aggregates; it has an activation energy of 22 +/- 5 kcal/mol; it is not related to a slow reaction in unfolding and thus probably not controlled by the cis-trans isomerization of X-Pro peptide bonds; its rate is inversely proportional to the solvent viscosity. It seems as if this reaction is limited by the mutual arrangement of the regions that have folded rapidly and independently. It is proposed that the mechanism where a fast folding of domains is followed by a slow pairing of folded domains could be generalized to other long chains composed of several domains; such a slow pairing of folded domains would correspond to a rate-limiting process specific to the renaturation of large proteins. The reappearance of the dehydrogenase activity measures the formation of a dimeric species. The dimerization can occur only after each chain has reached its "native" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
D001225:Aspartokinase Homoserine Dehydrogenase / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D009097:Multienzyme Complexes; D001225:Aspartokinase Homoserine Dehydrogenase; D019791:Guanidine