Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12.

Lunn, Charles A.; Kathju, Sandeep; Wallace, B J; Kushner, Sidney R.; Pigiet, Vincent

doi:10.1016/s0021-9258(18)90987-7

Cited by 121 publications

(56 citation statements)

References 26 publications

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“…The Trx-tagged fusion proteins were expressed successfully at good rates. Trx-based gene fusion expression systems have proven useful for the convenient expression and puri¢cation of weakly soluble proteins [27,47]. There are some examples of successful recombinant expression of an amyloid polypeptide as fusion construct with a soluble protein moiety.…”

Section: Discussionmentioning

confidence: 99%

Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)

Krampert

Bernhagen

Schmucker

et al. 2000

Chemistry & Biology

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Section: Discussionmentioning

confidence: 99%

Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)

Krampert

Bernhagen

Schmucker

et al. 2000

Chemistry & Biology

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“…E. coli thioredoxin (TR, 40 μmol/min/mg) and thioredoxin reductase (TRR, 1800 μmol/min/mg) were prepared as previously described. , Fluorotyrosines were synthesized enzymatically from pyruvate, ammonia, and the corresponding fluorophenol with tyrosine phenol lyase as previously described . Assay buffer consists of 50 mM HEPES, 15 mM MgSO 4 , and 1 mM EDTA adjusted to the specified pH.…”

Section: Methodsmentioning

confidence: 99%

Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase

2016

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Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multi-step proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ~ 35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask kinetic examination of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [ReI] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET) and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively “tight” subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits.

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“…[5- 3 H]Cytidine 5′-diphosphate sodium salt hydrate ([5- 3 H]CDP) was purchased from ViTrax (Placentia, CA), Tricarbonyl(1,10-phenanthroline)(4-bromomethyl-pyridine)rhenium(I) hexafluorophosphate ([Re I ]-Br) was available from a previous study , C 268 S/C 305 S/S 355 C/Y 356 W-β 2 and Y 356 W-β 2 were generated by site-directed mutagenesis using the primers described in the Supporting Information and expressed and purified as previously reported for related photoβ 2 variants . All photoβ 2 s were reduced with hydroxyurea prior to measurements to eliminate the native tyrosyl radical cofactor.…”

Section: Methodsmentioning

confidence: 99%

“…15,16 C 268 S/C 305 S/ S 355 C/Y 356 W-β 2 and Y 356 W-β 2 were generated by site-directed mutagenesis using the primers described in the Supporting Information and expressed and purified as previously reported for related photoβ 2 variants 12. All photoβ 2 s were reduced with hydroxyurea prior to measurements to eliminate the native tyrosyl radical cofactor.…”

mentioning

confidence: 99%

Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase

et al. 2016

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The E. coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox-active amino acids (β-Y122 ⇌ β-Y356 ⇌ α-Y731 ⇌ α-Y730 ⇌ α-C439) spanning ~35 Å and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be phototriggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y356 within the α/β subunit interface proceeds by a stepwise ETPT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y731/Y730 in α. This construct displays a ~3-fold enhancement in photochemical yield of W• relative to F3Y• and a ~7-fold enhancement relative to Y•. Photogeneration of the W• radical occurs with a rate constant of 4.4 ± 0.2 × 105 s−1, which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y → W variant displays no enzymatic activity in the absence of light, photogeneration of W• within the subunit interface results in 20% activity for turnover relative to wt-RNR under the same conditions.

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Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12.

Cited by 121 publications

References 26 publications

Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)

Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)

Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase

Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase

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