Crystallization and preliminary crystallographic data for thioredoxin from Escherichia coli B

Holmgren, Arne; Söderberg, Bengt-Olof

doi:10.1016/0022-2836(70)90437-7

Cited by 37 publications

(15 citation statements)

References 13 publications

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“…A crystal structure of oxidized E. coli thioredoxin was obtained for crystals containing Cu(II) (Holmgren and S6derberg, 1970;Holmgren et al, 1975). These conditions were unsuitable for the formation of crystals of the reduced form (Holmgren and S6derberg, 1970), and no other conditions have been found under which crystallization of either form of thioredoxin occurred.…”

Section: Introductionmentioning

confidence: 99%

“…These conditions were unsuitable for the formation of crystals of the reduced form (Holmgren and S6derberg, 1970), and no other conditions have been found under which crystallization of either form of thioredoxin occurred. A solution structure of the reduced form of E. coli thioredoxin was therefore obtained from ~H NMR data (Dyson et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

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Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

et al. 1994

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As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherichia coli thioredoxin (M(r) 11,700), we have analyzed the NMR data obtained for the two proteins under identical conditions. The complete aliphatic 13C assignments for both oxidized and reduced thioredoxin are reported. Correlations previously noted between 13C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C beta and C gamma shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys32-Gly-Pro-Cys35 sequence causes changes in the 1H, 15N and 13C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence. Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp28 and Trp31). These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

et al. 1994

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“…Crystallization of E. coli thioredoxin-S2 from alcoholic solution was found to be absolutely dependent upon the extraneous addition of cupric ions (10 The f-values refer to the average root mean squares of the scattering factors of each heavy atom derivative. The E-values represent the root mean square lack of closure errors of the phase triangles.…”

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confidence: 99%

Three-dimensional structure of Escherichia coli thioredoxin-S2 to 2.8 A resolution.

Holmgren¹,

Söderberg²,

Eklund³

et al. 1975

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

412

241

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The three-dimensional structure of the electron transport protein thioredoxin-S2 from E. coli has been determined from a 2.8 A resolution electron density map. The molecule is built up of a central core of three parallel and two antiparallel strands of pleated sheet surrounded by four helices. The residues involved in the active center 14-membered disulfide ring of thioredoxin form a protrusion between one of the helices and the middle strand of the pleated sheet. This region of the molecule, comprising two parallel strands joined by the protrusion and a helix, is structurally very similar to corresponding functionally important regions in the nucleotide-binding domains of flavodoxin and the dehydrogenases. The molecule has about 75% of the residues in well-defined secondary structures. The structure indicates that the carboxy-terminal third of the molecule forms an independent folding unit consisting of two strands of antiparallel pleated sheet and a terminal ahelix. This agrees with the noncovalent reconstitution experiments from thioredoxin peptide fragments. Thioredoxin is an example of a protein with the active center located on a protrusion rather than in a cleft, thus demonstrating the existence of male proteins.Thioredoxin is a low-molecular-weight protein which transports electrons via an oxidation-reduction active disulfide (1,2). Together with the FAD-containing enzyme, thioredoxin reductase, it forms a cyclic electron transport system (thioredoxin system) which makes the reducing power of NADPH available for reductions (3) (see Fig. 1.). Thioredoxin seems to be a universal component of all living cells capable of replicating DNA, since it is involved in the synthesis of deoxyribonucleotides (3, 4).Thioredoxin from Escherichia coli B has the following properties. It is an acidic protein, (isoelectric point 4.5) containing 108 amino-acid residues of known sequence (2) and is devoid of metals and cofactors. The oxidation-reduction disulfide bridge is formed from Cys-32 and Cys-35 spaced by Gly-33 and Pro-34, thereby forming a 14-membered disulfide ring (2). A localized conformational change accompanies reduction of thioredoxin-S2 to thioredoxin-(SH)2 (5) which drastically changes the fluorescence emission of 7). Noncovalent reconstitution of the thioredoxin molecule from two sets of peptide fragments obtained by selective cleavage at the single arginine or methionine (_Met-37) residues has been obtained (8,9). This suggests a favorable equilibrium of nucleated folding of the peptide fragments into their native format as found in thioredoxin (9).Crystallization of E. coli thioredoxin-S2 from alcoholic solution was found to be absolutely dependent upon the extraneous addition of cupric ions (10).

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“…It has been found in several experiments, not detailed here, in our and in other laboratories [4] that thioredoxins including authentic E. coli thioredoxin can elute from calibrated gel chromatography column in unexpected fractions, usually corresponding to a molecular mass several thousand daltons higher, depending upon the type of gel matrix, ionic strength, and redox state. (It is tempting to speculate that such anomalous behaviour is due to the specific protein folding of thioredoxins with a protruding, active-site chain segment [24]). In contrast, thioredoxins fit the normal, linear molecular weight scale when analyzed on SDS or ureacontaining polyacrylamidegels, calibrated with commonly used marker proteins plus E. coli thioredoxin of known molecular weight.…”

Section: Drtcmiination Of Molecular Size Qf Thioredoxirismentioning

confidence: 99%

Plant Seeds Contain Several Thioredoxins of Regular Size

1983

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Thioredoxin systems composed of several thioredoxin isoproteins and a NADPH : thioredoxin reductase are contained in the albumin-globulin fraction of wheat and soy-bean seed proteins. Two wheat thioredoxins I and I1 were separated on CM-cellulose whereas soy-bean extracts could be resolved into three thioredoxins I, 11, and I11 on DEAE-cellulose. These proteins were purified to apparent homogeneity and were shown by sodium dodecylsulfate/polyacrylamide gel electrophoresis to possess the molecular weight M, z 12000 typical of the single bacterial and animal thioredoxin. In contrast, gel filtration runs may yield erroneous estimates of thioredoxin molecular weights. The seed thioredoxins can serve as ribonucleotide reductase (Escherichia coli) substrates. They stimulate spinach NADP: malate dehydrogenase but are inactive towards chloroplast fructose-bisphosphatase. These results demonstrate that the number of thioredoxins in nongreen plant tissues approaches that of leaves; additional explanations must therefore be sought for the multiple thioredoxin profiles of plants besides diversification for light-dependent and light-independent functions.Plants are known to contain several protein fractions with thioredoxin activity [l -51. It is an open question whether this is so because in addition to established thioredoxin functions like hydrogen transfer for deoxyribonucleotide synthesis, sulfate, or protein disulfide reduction there are extra, specific thioredoxins that participate in light-affected chloroplast reactions, or whether one observes the not uncommon case of isoproteins having common origin and basically similar, although modulated reactivities. Previously reported properties of plant thioredoxins appeared to support the first view, and the proteins (especially those isolated from leaves) have been grouped into ones activating fructose-bisphosphatase ('f' type), others that preferentially stimulate NADP: malate dehydrogenase ('m' type), and 'c' thioredoxins of presumed cytoplasmic origin [I]. Such typification bears some arbitrariness as other enzymes like ribonucleotide reductases [6, 71, glutamine synthetase [28] or NADP: glyceraldehyde-3-phosphate dehydrogenase [30] are also activated but are not usually measured for practical reasons. In fact, the more or less unlimited exchangeability in v i m of many bacterial. animal, and plant thioredoxins in completely unrelated enzyme systems [6, 71 suggests that all these small -SH proteins are closely related and not highly specialized. The long-known existence of two thioredoxin isoproteins in yeast [S] and the preliminary characterization of more than one thioredoxin in photosynthetic bacteria and cyanobacteria [9, 101 also indicate that thioredoxin multiplicity is not restricted to eukaryotic plant cells, nor to photosynthetic organisms at all.

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Crystallization and preliminary crystallographic data for thioredoxin from Escherichia coli B

Cited by 37 publications

References 13 publications

Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

Three-dimensional structure of Escherichia coli thioredoxin-S2 to 2.8 A resolution.

Plant Seeds Contain Several Thioredoxins of Regular Size

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