Stepwise inactivation of Escherichia coli aspartokinase-homoserine dehydrogenase I

Müller, K; Garel, Jean‐Renaud

doi:10.1021/bi00299a010

Cited by 9 publications

(11 citation statements)

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“…As in the case of many proteins (Jaenicke, 1984), this extent of reactivation depends markedly on the concentration of AK-HDH chains at which renaturation occurs: up to about 5 nM all the activity can be regained, between 5 and 50 nM this extent decreases, and above 50 nM almost no activity can be recovered (Figure 4). This is usually interpreted as resulting from a kinetic competition between folding and aggregation at some branch point X (Jaenicke, 1982(Jaenicke, , 1984Garel et al, 1984;Müller & Garel, 1984b):…”

Section: The Slow Reactivationmentioning

confidence: 99%

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

Vaucheret¹,

Signon²,

Bras³

et al. 1987

Biochemistry

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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)

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Section: The Slow Reactivationmentioning

confidence: 99%

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

Vaucheret¹,

Signon²,

Bras³

et al. 1987

Biochemistry

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“…One of the major complications encountered in the study of the unfolding-refolding process of oligomeric proteins is the irreversible aggregation of partially folded species (Jaenicke, 1982); in the case of AK-HDH I, quantitative renaturation can only be achieved if the protein concentration is maintained at a low value (Garel & Dautry-Varsat, 1980a). The preceding paper (Müller & Garel, 1984) shows that a marked change in the solubility of AK-HDH I occurs when the Gdn-HCl concentration is raised above 0.2 M, probably as a result of the protein dissociation. Increasing the Gdn-HCl concentration above 0.2 M accelerates this aggregation [see Figure 1 of Müller & Garel (1984)] because partial unfolding is favored.…”

Section: Resultsmentioning

confidence: 99%

“…Materials. Unless otherwise stated, all materials used in this study are the same as those given in the preceding paper (Müller & Garel, 1984). Buffer solutions were prepared with quartz bidistilled water in the case of CD measurements; water which had been pyrolyzed after deionization was utilized in fluorescence studies: the traces of fluorescent organic molecules present in water are destroyed more satisfactorily by this high-temperature treatment than by any other method.…”

Section: Methodsmentioning

confidence: 99%

“…The existence of a stable monomeric and folded intermediate indicates that the tertiary interactions have a major contribution to the .^^.spartokinase-homoserine dehydrogenase I (AK-HDH I)1 from Escherichia coli'vs a bifunctional protein composed of four identical polypeptide chains (Afr 89000) each carrying the sites involved in both activities; the kinase activity corresponds to the amino-terminal part of each chain and the dehydrogenase activity to the carboxy-terminal part (Veron et al, 1972;Théze & Saint-Girons, 1974). The native state of AK-HDH I has a tetrameric structure, but two different lines of evidence suggest that functional (and therefore folded) species can exist as monomers in the absence of any subunit interactions: (a) limited proteolysis of native AK-HDH I yields monofunctional fragments which are monomeric and active (Fazel et al, 1983); (b) the kinetics of deactivation and reactivation of AK-HDH I are compatible with the existence of a monomeric intermediate possessing the kinase activity (Garel & Dautry-Varsat, 1980a; Müller & Garel, 1984).…”

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confidence: 99%

“…In the case of the entire AK-HDH I chain, folded monomers have only been observed as transient intermediates in deactivation or reactivation (Garel & Dautry-Varsat, 1980a; Müller & Garel, 1984); also, the lability of the monomeric active fragments obtained by proteolysis indicates that they could only correspond to metastable species (Fazel et al, 1983). In addition, only activity measurements have been used to establish the formation of a folded structure in a given segment of the chain; it is obvious that such a functional definition of a folded state is strongly biased since large segments of a polypeptide chain can take a stable, specific, and highly organized conformation without being active.…”

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

Müller¹,

Garel²

1984

Biochemistry

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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.

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Proteinfaltung und Proteinassoziation

Jaenicke

1984

Angewandte Chemie

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Die Biosynthese von Proteinen führt zu linearen Kettenmolekülen mit streng definierter Reihenfolge der Aminosäurebausteine. Diese ist aufgrund des genetischen Codes auf der Ebene der Nucleinsäure durch die Nucleotidsequenz des zugehörigen Gens festgelegt. Denaturierungs‐Renaturierungs‐Experimente legen die These nahe, die in der Polypeptidkette vorgegebene eindimensionale Information determiniere die dreidimensionale Struktur vollständig. Demnach sollte ein „Faltungscode”︁ existieren, der das spontane Zustandekommen der thermodynamisch stabilen „nativen”︁ Struktur bestimmt. Der Prozeß der Faltung (in vivo wie in vitro) ist trotz der astronomisch großen Zahl möglicher Konformationen ein schneller Vorgang. Der Faltungsmechanismus muß daher kinetisch kontrolliert sein. Die Frage, ob die native Struktur letztlich ein „lokales”︁ oder das „globale”︁ Minimum der Energiehyperfläche besetzt, bleibt offen. Oberhalb einer kritischen Molekülgröße liegen Proteine als Molekülassoziate vor. Die Einheitlichkeit der „Quartärstruktur”︁ setzt eine enge Korrelation von Faltung und Assoziation sowie Spezifität voraus; beides wird durch Rekonstitutionsexperimente in vitro bestätigt.

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Stepwise inactivation of Escherichia coli aspartokinase-homoserine dehydrogenase I

Cited by 9 publications

References 23 publications

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

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

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

Proteinfaltung und Proteinassoziation

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