Differences in hydrogen exchange behavior between the oxidized and reduced forms of <i>Escherichia coli</i> thioredoxin

Kaminsky, Stephen M.; Richards, Frederic M.

doi:10.1002/pro.5560010103

Cited by 19 publications

(13 citation statements)

References 35 publications

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“…Because the first &strand also has a large positive free energy of solvation and it is only 10 A from the active site, it is possible that cooperative changes occur at this site as well. These two regions correspond closely to the peptides that undergo a change in amide proton exchange rates upon reduction of thioredoxin (Kaminsky & Richards, 1992). The adiabatic compressibility of reduced thioredoxin is in the middle of the published range of -1 .O to t 1 2 .…”

Section: Sm Kaminsky and F M Richardsmentioning

confidence: 62%

“…In NMR experiments Hiraoki et al (1988) and Dyson et al (1990) found redox-sensitive changes, including hydrogen exchange properties, in the region of the SS bond. In the previous paper Kaminsky and Richards (1992) have corroborated that there is a small group of amide protons whose exchange behavior is dramatically altered by the redox state. These appear to occur in two small nonadjacent regions, the disulfide loop and the first 6strand.…”

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confidence: 78%

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Reduction of thioredoxin significantly decreases its partial specific volume and adiabatic compressibility

Kaminsky

Richards

1992

Protein Science

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The partial specific volume and adiabatic compressibility were determined at several temperatures for oxidized and reduced Escherichia coli thioredoxin. Oxidized thioredoxin had a partial specific volume of 0.785-0.809 mL/g at the observed upper limit for all proteins whereas the partial specific volume of reduced thioredoxin was 0.745-0.755 mL/g, a value in the range found for a majority of proteins. The adiabatic compressibility of oxidized thioredoxin was also much larger (9.8-18 x 10(-12) cm2 dyne-1) than that of the reduced protein (3.8-7.3 x 10(-12)). Apart from the region immediately around the small disulfide loop, the structures of the oxidized (X-ray, crystal) and reduced protein (nuclear magnetic resonance, solution) are reported to be very similar. It would appear that alterations in the solvent layer in contact with the protein surface must play a major role in producing these large changes in the apparent specific volumes and compressibilities in this system. Some activities of thioredoxin require the reduced structure but are not electron transfer reactions. The large changes in physical parameters reported here suggest the possibility of a reversible metabolic control function for the SS bond.

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Section: Sm Kaminsky and F M Richardsmentioning

confidence: 62%

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confidence: 78%

Reduction of thioredoxin significantly decreases its partial specific volume and adiabatic compressibility

Kaminsky

Richards

1992

Protein Science

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“…Recently, Dyson et al (1994) confirmed that the overall structure of the tnutant [Ser32, Ser35lTrx of E. coli thioredoxiii is very similar to that of the wild-type thioredoxin. Two regions are significantly more mobile than the average: the Nterminus and the active site (Kaminsky and Richards, 1992a;Stone et al, 1993). Substitution of glycine for serine at position 74 does alter the backbone structure.…”

Section: Discussionmentioning

confidence: 99%

“…New structural inforimtion oht;iined frorri models constructcd from tliioi-cdoxins of several oi-ganisnis, bascd on NMK sludies (Dyson ct al., 1989; Forinan-Kay e l al., 1989; Kaminsky and Richards, 19922;Stone et ul., 1993;Jeng el al., 1994) and o n the crysrallographic structure of E. coli thioredoxin, indicates that 311 protcins have similar throc-dimcnsiunal struclures dcspite the large variation in arnino acid sequences (Ekliind ct al., 1091). Two nun-adjacent regions, either in the linear sequence or in thrcc-dimensional space, exhibit changes upon reduction : the active site, residues 32-35, and a region within residues 1 -I3 at the N-terminal end (Kaminsky and Richards, 1992a;Stone Thioredoxin from Rho$ohrrcfcr sp/iwmi&s is similai-to E. coli thiorediixin and exhibits 47 % arnino acid identity to this protein. We have previously shown that the expression of R. S/JhLWI'Oim't'S thioredox in from ii recoinhiiiant plasmid, i ntroduccd into ii mutant of E. roli lacking thioredoxin, can restorc thc growth 01' phagc T7 (Pille ct al., 1990).…”

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confidence: 99%

The Gly74→Ser and Ser3→Ala Mutations in Rhodobacter sphaeroides Y Thioredoxin

Pille

Assemat

Breton

et al. 1996

European Journal of Biochemistry

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In this study, we report the effects of two different substitutions in Rhodohacter ,vphmroidf,.s thioredoxin on two regions of the protcin : the N-terminus end and thc hydrophobic area implicated in protein/ protein interactions. We have produced by site-dirccterl mutagcnesis R. ,sphnevoides thiorcdoxin single and double mutants in which the glycine residue at position 74 is changed to a scrine and the serinc :it position 3 is changed to an alanine; the three mutant proteins have been purified. Thc two substitutions are not equivalent. Substitution of serine by aliinine increascd the pl from 5.2 to 6.1 ; this pl value was the same in the double-mutated protein, which dcmonstrates the presence of a local cotiformational change. 111 vivo studies showed that the Gly74-.Ser substitution completely prevented phage T3/7 growth whereas the Ser34Ala substitution had no effect. This finding was corroborated by the large decrease ( IOO-fold) of polymerase activity for the double niutant in the in vilm measurement of phage T7 DNA polymcrase activity with the corresponding pure proteins. Although trxirginal (within a factor of two), the effects of the two substitutions on the calalytic activities of the thioredoxin reductase rciiction con finned their difference. Substitution of serine by alanine had no effect on the K,,, and resulted in iin improvement in the catalytic efficiency. In contrast, the second substitution increased the K,,, value, without improving the catalytic efficiency. The following can be concludcd (a) glyciiie74 of R. sp/iwroidt<.s thiaredoxin has a direct role in the binding of T7 gene 5 protein iind thc hydrophobic area of thioredoxin: (b) the Nterminus plays a role in tnaintaining the conforniiitional integrity of the active site; (c) the flcxibility of Gly74 in the hydrophobic region involved in protcin/protein interaction is the operative factor in the case of the activity of thioredoxin in the T7 DNA polymcrase.Keywords: thioredoxin; T7 DNA polymerase ; site-directed niutagenesis; kinetic constants; stt-iictui-c/ fuiiction relationships.Thioredoxin i s a small, heat-stable rcdox protein with the active-center amino acid sequence -Trp-Cys-C;ly-Pro-Cyshighly conserved in all but 1 of the 34 sequences currently rcported (Swiss-Prot data bank). This protein seetiis to be present in all living organisms from archaebactcria to humans and hiis now heen characterized from a wide variety of procaryotic iind eucaryotic cells (Holmgren, 1985 ;Gleason and Holmgren, 1988;Eklund et al., 1991;Pille, 1992). Thioredoxin has diverse functions in the cell, several of which depcnd on redox activity via thiol-disulfidc interchange reactions. In addition to thcir redox functions, some members of this class have structural roles in coliphages such as f l and M i 3 (Russel and Model, 198s; Litn et al., 1986;Russel, 1991) and as a subunit of T7 DNA polymerasc (Modrich and Richardson, 1975). The structurc of the reduced form is required for the function of thioredoxin in coliphages although the redox property is not otherw...

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“…Oxidation of the NTD slightly increases stability and causes increased internal fluctuations (wider well) of rapidly interconverting states on an intermediate timescale (rougher well). Oxidation of Ec Trx increases stability (49) and decreases the conformational heterogeneity (51, 52) (narrower well) without large changes in intermediate timescale motions (50) (similar roughness).…”

Section: Figures and Tablesmentioning

confidence: 93%

Redox-Dependent Dynamics of a Dual Thioredoxin Fold Protein: Evolution of Specialized Folds

et al. 2009

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An enzyme system protecting bacteria from oxidative stress includes the flavoprotein AhpF and the peroxiredoxin AhpC. The N-terminal domain of AhpF (NTD), with two fused thioredoxin (Trx) folds, belongs to the hyperthermophilic protein disulfide oxidoreductase family. The NTD is distinct in that it contains a redox active a fold with a CxxC sequence and a redox inactive b fold that has lost the CxxC motif. Here we characterize the stability, the 15N backbone relaxation, and the hydrogen deuterium exchange properties of reduced (NTD-(SH)2) and oxidized (NTD-S2) NTD from Salmonella typhimurium. While both NTD-(SH)2 and NTD-S2 show similar equilibrium unfolding transitions and order parameters, Rex relaxation terms are quite distinct with considerably more intermediate timescale motions in NTD-S2. Hydrogen exchange protection factors show that the slow exchanging core corresponds to residues in the b fold in both NTD-(SH)2 and NTD-S2. Interestingly, folded state dynamic fluctuations in the catalytic a fold are significantly increased for residues in NTD-S2 compared to NTD-(SH)2. Taken together, these data demonstrate that oxidation of the active site disulfide does not significantly increase stability but results in a dramatic increase in conformational heterogeneity in residues primarily in the redox active a fold. Differences in dynamics between the two folds of the NTD suggest that each evolved a specialized function which, in the a fold, couples redox state to internal motions which may enhance catalysis and specificity, and in the b fold, provides a redox insensitive stable core.

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Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin

Cited by 19 publications

References 35 publications

Reduction of thioredoxin significantly decreases its partial specific volume and adiabatic compressibility

Reduction of thioredoxin significantly decreases its partial specific volume and adiabatic compressibility

The Gly74→Ser and Ser3→Ala Mutations in Rhodobacter sphaeroides Y Thioredoxin

Redox-Dependent Dynamics of a Dual Thioredoxin Fold Protein: Evolution of Specialized Folds

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