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

Abstract

In the presence of guanidine hydrochloride concentrations above 2 M, aspartokinase-homoserine dehydrogenase I 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 between 3 and 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 two complementary nonoverlapping fragments: a kinase fragment corresponding to the N-terminal third and a dehydrogenase fragment corresponding to the C-terminal two-thirds 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: indeed, an equimolar mixture of these two fragments and the intact chain would give about the same results. This indicates that folding of the kinase and dehydrogenase regions occurs independent ly with a single subunit of the entire protein.

Mechanism of renaturation of a large protein, aspartokinase-homoserine dehydrogenase

“…A Faster Reaction Corresponding to the Major Folding Process Is Followed by a Slower Isomerization That Restores the Kinase Activity. In 3 M Gdn-HCl, AK-HDH exists as a stable (partially) folded monomeric species as seen from its fluorescence and circular dichroism properties (Garel et al, 1984;Müller & Garel, 1984a). The formation of this folded monomer from the unfolded chain has a half-life of 6.5 min in 3 M Gdn-HCl and of less than 0.5 min in 2 M Gdn-HCl (Müller & Garel, 1984a).…”

“…In 3 M Gdn-HCl, AK-HDH exists as a stable (partially) folded monomeric species as seen from its fluorescence and circular dichroism properties (Garel et al, 1984;Müller & Garel, 1984a). The formation of this folded monomer from the unfolded chain has a half-life of 6.5 min in 3 M Gdn-HCl and of less than 0.5 min in 2 M Gdn-HCl (Müller & Garel, 1984a). Upon dilution of AK-HDH from 6 M to less than 0.1 M Gdn-HCl, there is a fluorescence change too rapid to be measured after manual mixing; this holds even at lower temperature (down to 5 °C) or in the presence of glycerol [up to 30% (v/v)].…”

“…(i) Many large polypeptide chains appear as composed of several compact regions, which are supposed to correspond to independent folding segments (Wetlaufer, 1973;Schulz & Schirmer, 1979; Janin & Wodak, 1983); this is indeed the case with AK-HDH in which three compact regions exist (Fazel et al, 1983) and for which the presence of independently folding units has been shown (Dautry-Varsat & Garel, 1981; Müller & Garel, 1984a).…”

“…e renaturation of an oligomeric protein from its unfolded and separated chains implies a mixture of folding and association reactions (Jaenicke, 1982(Jaenicke, , 1984Jaenicke & Rudolph, 1980). In the case of aspartokinase-homoserine dehydrogenase (AK-HDH),1 a tetrameric and bifunctional enzyme, the renaturation process could be decomposed into a succession of several steps (Garel & Dautry-Varsat, 1980a,b;Dautry-Varsat & Garel, 1981; Müller & Garel, 1984a): (i) a polypeptide chain acquires some organized structure, as seen from its fluorescence and circular dichroism, and forms a stable (partially) folded monomeric species; (ii) this (partially) folded species further isomerizes to yield an intermediate with full kinase activity and still a monomeric structure; (iii) two folded monomers then associate into a dimeric species that possesses a normal dehydrogenase activity; (iv) finally, two dimers as-tThis work was supported by the Centre National de la Recherche (UA 1129), the Instituí Pasteur, and the Université Paris 6 (UER 58).…”

“…Both fragments can resume their functional structure after complete unfolding. The comparison between the kinetics of reactivation of the fragments and that of the entire chain shows that neither the mechanism nor the rate of renaturation is markedly affected by the removal of 30-40% at either end of the chain (Dautry-Varsat & Garel, 1981; Müller & Garel, 1984a). It seems then that, during the renaturation of AK-HDH, several segments along the polypeptide chain behave as independent folding units.…”