Independent folding regions in aspartokinase-homoserine dehydrogenase

Dautry‐Varsat, Alice; Garel, Jean‐Renaud

doi:10.1021/bi00508a056

Cited by 42 publications

(14 citation statements)

References 26 publications

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“…The succession of events that can be proposed from this and previous work (Garel & Dautry-Varsat, 1980a,b;Dautry-Varsat & Garel, 1981;Müller & Garel, 1984a) for the process producing native AK-HDH from its unfolded and separated chains can be compared to the results obtained with other domain-containing proteins:…”

Section: Discussionmentioning

confidence: 94%

“…Most experimental methods have been described previously: the preparation of AK-HDH and of its fragments, the assays for the kinase and dehydrogenase activities, the denaturation and renaturation procedures, the composition of the various buffers, the kinetic analysis, etc. (Garel & Dautry-Varsat, 1980a; Dautry-Varsat & Garel, 1981;Müller & Garel, 1984a,b). The viscosity of the solvent was changed by adding either glycerol or sucrose, and the increase in viscosity was taken from the tabulated values given in the Handbook of Chemistry and Physics (1973).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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: Discussionmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Vaucheret¹,

Signon²,

Bras³

et al. 1987

Biochemistry

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“…Previous studies with spontaneously refolded proteins, e.g. tryptophan synthetase (21, 22), dihydrofolate reductase (23), and aspartokinasehomoserine-dehydrogenase (24,25), show that individual domains in the entire polypeptide chain refold differentially and can be expressed as soluble functional units, suggesting that the domain alone is a folding unit. Co-translational independent domain folding in eukaryotes has been recently demonstrated using a Ras-dihydrofolate reductase fusion polypeptide (26).…”

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confidence: 99%

Mechanisms for GroEL/GroES-mediated Folding of a Large 86-kDa Fusion Polypeptide in Vitro

Huang¹,

Chuang

1999

Journal of Biological Chemistry

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Our understanding of mechanisms for GroEL/GroESassisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the ␣ subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain ␣-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-␣, when co-expressed with the ␤ subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-␣) 2 ␤ 2 structure, similar to the ␣ 2 ␤ 2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/ GroES and Mg 2؉ -ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M ؊1 s ؊1 , analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-␣ fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the ␣ moiety on the same chain with rate constants of 1.9 ؋ 10 ؊3 s ؊1 and 2.95 ؋ 10 ؊4 s ؊1 , respectively. This explained the occurrence of an MBP-␣⅐GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the ␣⅐GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His) 6 -␣ polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP␣⅐GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES.

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“…However, few direct measurements of the activity of separate domains during the folding of multidomain proteins have been described and these are based mostly on the separate folding of proteolytically derived fragments (17)(18)(19). PE and the PE-derived single-chain immunotoxins can be analyzed by activity assays to determine whether individual domains fold independently from each other.…”

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confidence: 99%

Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections.

Brinkmann

Büchner²,

Pastan³

1992

Proc. Natl. Acad. Sci. U.S.A.

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We have studied the refolding of completely unfolded and reduced Pseudomonas exotoxin (PE) and of recombinant single-chain immunotoxins made with monoclonal antibody B3 that are composed of a heavy-chain variable region connected by a flexible linker to the corresponding light-chain variable region (Fv), which is in turn fused to a truncated form of PE. We have found by direct activity assays that different functional domains of these multf onal proteins fold independently with different kinetics. The ADPribosylation domain of PE and of the recombinant immuntoxin fold rapidly, whereas the assembly of the binding and/or translocation domains is regained more slowly. The complete refolding of native PE occurs more rapidly than the refolding of the recombinant immuoxins. To determine the influence of the connector region between the B3(Fv) moiety and the toxin on the folding process of the recombinant immunotoxin B3(Fv)-PE38KDEL, we have made two different mutations in the peptide that connects the single-chain Fv domain to domain II of PE. These molecules show different folding kinetics, differences in their propensity to aggregate, and different yields of correctly folded molecules. A mutation that decreases aggregation increases the rate of formation and the yield of active immunotoxin molecules.Pseudomonas exotoxin (PE) is a 66-kDa multifunctional protein that kills eukaryotic cells by ADP ribosylating elongation factor 2 (1-3). PE consists of three major structural domains, each having at least one distinct function. Domain Ia is responsible for cell binding, and domain II mediates translocation of the ADP-ribosylating portion (domain III) of PE into the cytosol of the cell (4). In addition, a small sequence at the carboxyl end of PE delivers the molecule to an intracellular compartment from which translocation occurs (5, 6). Deletion or inactivation of any of these domains results in loss of cytotoxicity. It is possible to substitute for

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Independent folding regions in aspartokinase-homoserine dehydrogenase

Cited by 42 publications

References 26 publications

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

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

Mechanisms for GroEL/GroES-mediated Folding of a Large 86-kDa Fusion Polypeptide in Vitro

Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections.

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