Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin

Kelley, Robert F.; Shalongo, William; Jagannadham, Medicherla V.; Stellwagen, Earle

doi:10.1021/bi00379a029

Cited by 55 publications

(57 citation statements)

References 15 publications

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“…Assuming an approximately constant solvent-accessible surface for the folded protein regardless of redox state, this suggests that the unfolded, oxidized form of the protein retains some residual structure. Denaturant m G values obtained from other oxidoreductases with the active-site sequence CX 1 X 2 C and mutants thereof also follow this trend with very few exceptions (9,39,40). Perhaps the inability of the oxidized form to fully unfold may be a factor in determining the redox potential of these proteins.…”

Section: Discussionmentioning

confidence: 65%

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

Åslund

Berndt

Holmgren

1997

Journal of Biological Chemistry

381

336

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Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2 C). Standard state redox potential (E°) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E° resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reversephase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pairwise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E° values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E° ‫؍‬ ؊233 and ؊198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E° values from a number of proteins ranging from ؊270 mV for E. coli Trx to ؊124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as ؊240 mV. Determining redox potentials through direct proteinprotein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.Glutaredoxin (Grx1) 1 was discovered as a GSH-dependent hydrogen donor for ribonucleotide reductase in Escherichia coli mutants lacking the first identified electron donor, thioredoxin (1). The presence of an additional hydrogen donor system for ribonucleotide reductase was postulated since a double mutant lacking both Grx1 and Trx was viable (2). The search for this third hydrogen donor system resulted in the isolation of two additional glutaredoxins in E. coli, Grx2 and Grx3 (3). The recent structural characterization of Grx3 showed that in addition to the 33% amino acid sequence identity with Grx1, the two proteins have highly conserved secondary structure elements and overall fold (4). However, despite Grx1 and Grx3 being closely related 9-kDa redox proteins with identical active-site sequences (CPYC), Grx3 exhibits only a fraction of the activity of Grx1 as a reductant of ribonucleotide reductase (NrdAB and NrdEF; Refs. 3 and 5) or of insulin disulfides (4). ...

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Section: Discussionmentioning

confidence: 65%

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

Åslund

Berndt

Holmgren

1997

Journal of Biological Chemistry

381

336

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“…Although the values of C m and ΔG 0 for this mutant are similar to that of reduced wild-type thioredoxin, 49 it has been confirmed by MS and thiolspecific labeling studies that the active site disulfides are fully oxidized in this dimer. Moreover, since the F102C and A105C mutations are far from the active site (N15 Å), there is no obvious reason why mutations at these sites should affect the redox potential of the active site disulfides between residues 32 and 35 in the thioredoxin monomer.…”

Section: Chemical and Thermal Stabilities Of The Dimersmentioning

confidence: 67%

Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

Das

Kobayashi

Yamada

et al. 2007

Journal of Molecular Biology

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“…a Swint and Robertson, 1993;' O'Neil et al, 1995;Hurle et al, 1990;Viguera et al, 1994; e Jackson et al, 1993;'Akke and Forsen, 1990;Khorasanizadeh et al, 1993;Wintrode et al, 1994;Scholtz, 1995;J Agashe and Udgaonkar, 1995;Lim et al, 1992; ' Marqusee and Sauer, 1994;McLendon and Smith, 1978;Hagihara et al, 1994; O Privalov and Gill, 1988;P Pace et al, 1990;Shirley et al, 1992;Yu et al, 1994; ' Bowie and Sauer, 1989; ' Egan et al, 1993;Ramdas et al, 1986;"Bryant et al, 1985; " Cohen and Pielak, 1994;x Kelley et al, 1987;Santor0 and Bolen, 1992;'Clarke and Fersht, 1993; aa Pace et al, 1992;" Griko et al, 1994; cc Greene and Pace, 1974;dd Munson et al, 1994;Filimonov et al, 1993; f f Saito and Wada, 1983; gg Ahmad and Bigelow, 1982; hh Taniyama et al, 1992; I' Herning et al, 1992;'' Ropson et al, 1990; kk Shortle and Meeker, 1986;"Carra et al, 1994; mm Craig et al, 1987; "" Makhatadze et al, 1994;O0 De Young et al, 1993...…”

Section: Aasamentioning

confidence: 99%

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

1995

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Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (AC,), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and AC,, Keywords: denaturation; guanidine hydrochloride; heat capacity changes; m values; protein folding; protein stability; solvent-accessible surface area; urea It has been known for many years that proteins can be unfolded in aqueous solution by high concentrations of certain reagents such as guanidine hydrochloride or urea. Denaturation with these chemicals is one of the primary ways of measuring the conformational stability of proteins and comparing the stabilities of mutant proteins. The use of these two denaturants is extremely widespread (Pace, 1986), even though the exact nature of the molecular interaction of denaturant molecules with protein surfaces is not well understood. It is known from solubility and transfer experiments with model compounds that the interaction of urea and Gdn HCI with the constituent groups of proteins is more favorable than the interaction of those groups with water (Tanford, 1970). These denaturants alter the equi-~"

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Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin

Cited by 55 publications

References 15 publications

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

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