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

Vaucheret, Hervé; Signon, L.; Bras, Gisèle Le; Garel, Jean‐Renaud

doi:10.1021/bi00384a020

Cited by 42 publications

(29 citation statements)

References 40 publications

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“…In Kramers' theory (18) and in the diffusion-collision model (19,20) linear diffusion of two reactants or of two folding domains is considered. Domain-domain interactions and the association of subunits during folding are such linear diffusion processes, and indeed their time constants depend linearly on solvent viscosity (37)(38)(39)(40)(41)(42). The collapse of the extended polypeptide chain in the folding of a small protein (such as CspB) is probably a process of higher dimensionality than considered in Kramers' model (18,43), and this might lead to a nonlinear relationship between reaction time and solvent viscosity.…”

Section: Folding Of Cspb Depends Strongly On Solvent Viscositymentioning

confidence: 99%

Diffusion control in an elementary protein folding reaction

Jacob

Schindler

Balbach

et al. 1997

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

139

163

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The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N ª U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose. The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together. It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding. Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy. We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein. This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior.Most protein chains reach their native conformations during folding rapidly and with high precision, even though the native state is only marginally stable and even though an unfolded protein can adopt very many conformations. Often it is assumed that the folding process is so efficient, because it occurs in two distinct stages (1-4). In the first stage the extended protein chain collapses rapidly into a compact form (often called a molten globule), which is already native-like, but still loosely packed (4, 5). In the second, slow stage the protein chain rearranges to the native state, possibly by a restricted search through the compact conformations. This stage shows a high energy barrier and determines the overall rate of folding. Until recently it was assumed that the rapid formation of compact intermediates is a prerequisite for fast and efficient folding (2,(4)(5)(6)(7)(8).Several small proteins fold extremely fast within a millisecond or even less, but no partially structured intermediates could be detected in these folding reactions (9-17). This seems puzzling. Either these proteins do not follow the two-stage model in their folding, or the initial collapse is so specific that the second stage becomes extremely fast. Thus, the compact intermediate would not accumulate, and the diffusive collapse would become rate-limiting for the entire folding reaction. In this case folding should not follow a monoexponential time course. As a third possibility, chain compaction and crossing of the energy barrier could be tightly coupled in folding. Kramers (18) developed a kinetic model for reactions in which the reactants diffuse together in the rate-limiting step, and he found that the time constants of such processes should depend linearly on the viscosity of the medium. Kramers' theory was used by Karplus and Weaver (19,20) when they formulated the diffusion-collision model for protein folding. Folding reactions that are limited in rate by...

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Section: Folding Of Cspb Depends Strongly On Solvent Viscositymentioning

confidence: 99%

Diffusion control in an elementary protein folding reaction

Jacob

Schindler

Balbach

et al. 1997

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

139

163

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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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“…In the case of many large proteins, however, additional slow steps, such as domain pairing and/or association reactions occur R . Rudolph et al (Jaenicke, 1987;Teschner et al, 1987;Vaucheret et al, 1987;Chrunyk & Matthews, 1990) and can become rate limiting for the overall folding reactions.…”

Section: Resultsmentioning

confidence: 99%

“…The folding of large proteins is far less well characterized, as their unfolding is often only partially reversible (Jaenicke, 1987). In some cases, very slow reactions in the minutes to hours time region are observed, which are not caused by isomerization reactions of prolyl peptide bonds but rather involve diffusion processes of folded parts of the molecules (Rudolph et al, 1986;Teschner et al, 1987;Vaucheret et al, 1987;Chrunyk & Matthews, 1990).…”

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confidence: 99%

Reversible unfolding and refolding behavior of a monomeric aldolase from staphylococcus aureus

1992

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Thermal and GdmC1-induced unfolding transitions of aldolase from Stuphylococcus uureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a AGO value of -9 f 2 kJ/mol at 20 "C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 "C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of a//3 barrel proteins.Keywords: a//3 barrel; folding intermediates; prolyl isomerization; protein folding kinetics; two-state model The mechanism of protein folding has been the subject of intensive studies over the last years. Experimental approaches to the folding problem have mainly focused on the elucidation of folding pathways, the characterization of folding intermediates, and the investigation of the dominant forces in protein stability (for reviews see Jaenicke, 1987;Privalov & Gill, 1988;Kuwajima, 1989;Kim & Baldwin, 1990). It is now widely accepted that protein folding advances through a number of definite intermediate stages (Baldwin, 1990). In the millisecond time region, secondary structural elements and parts of the tertiary contacts are formed (Roder et al., 1988;Udgaonkar & Baldwin, 1988Kuwajima, 1989). Subsequent slow steps are often limited by cis trans isomerization reac- Abbreviations; Aldolase, fructose-l,6-bisphosphate aldolase (EC 4.1.2.13) from Stuphylococcus aureus; GdmC1, guanidinium chloride; N, native state of the protein; U, unfolded protein; UF and Us, fast and slow refolding molecules, respectively; AG, free energy of folding; a, reduced amplitude; X, apparent rate constant; 7, relaxation time.

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

Cited by 42 publications

References 40 publications

Diffusion control in an elementary protein folding reaction

Diffusion control in an elementary protein folding reaction

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

Reversible unfolding and refolding behavior of a monomeric aldolase from staphylococcus aureus

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