Streamlined purification and characterization of Pyrococcus furiosus rubredoxins with different N-terminal modifications by reversed-phase HPLC

Lin, Shaomin; He, Chunmao

doi:10.1016/j.ab.2021.114128

“…The expression and purification of the pf Rd variants were carried out similarly to that of wt- pf Rd, , but iron–sulfur clusters were only successfully constructed in pf Rd-C9A and pf Rd-C42A. The fact that the iron–sulfur center cannot be established in pf Rd-2CA and -3CA clearly indicates the importance of three sulfhydryl groups for iron coordination.…”

Section: Resultsmentioning

confidence: 99%

Development of Nonheme {FeNO}⁷ Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Lin

¹

,

He

²

2021

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Nitric oxide (NO) is an essential biological messenger, contributing a significant role in a diverse range of physiological processes. The light-controllable NO releasers are of great interest because of their potential as agents for NO-related research and therapeutics. Herein, we developed a pair of red-light-controllable NO releasers, pfRd-C9A-{FeNO}7 and pfRd-C42A-{FeNO}7 (pfRd = Pyrococcus furiosus rubredoxin), by constructing a nonheme {FeNO}7 center within the redesigned iron–sulfur protein scaffolds. While shown to be both air and thermally stable, these complexes are highly sensitive to red-light irradiation with temporal precision, which was confirmed by electron paramagnetic resonance spin trapping and Griess assay. The temporally controlled NO release from these complexes was also demonstrated in DNA cleavage assay. Overall, this study demonstrates that such a protein-based nonheme iron nitrosyl system could be a viable chemical tool for precise NO administration.

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“…The purified protein 12 was refolded and reconstituted with Fe 3 + ions as reported, [24] and the semi-synthetic protein was confirmed by its characteristic electronic absorption at 340 nm and 420 nm (Figure S7), which showed no obvious difference to the expressed protein. [24,25]…”

Section: Semi-synthesis Of Rubredoxin Through the Tcepl Strategymentioning

confidence: 99%

Protein Ligation and Labeling Enabled by a C‐Terminal Tetracysteine Tag

Mo

¹

,

Lin

²

,

Chen

³

et al. 2022

Angewandte Chemie

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The hydrazinolysis of S-cyanylated peptide provides an alternative way to afford protein αhydrazide, a key reagent used in native chemical ligation (NCL), without the aid of any inteins or enzymes. The currently used non-selective S-cyanylation, however, allows no other cysteine in the protein besides the one at the cleavage site. Herein, we report a regioselective Scyanylation and hydrazinolysis strategy achieved via the fusion of a tetracysteine tag to the C-terminal of the protein of interest. We term it tetracysteine enabled protein ligation (TCEPL). While highly selective, the strategy is applicable for proteins expressed as inclusion bodies, and this was showcased by the efficient semisynthesis of an iron-sulfur protein rubredoxin and the catalytic and hinge domains of matrix metalloprotease-14 (MMP-14) containing 207 amino acid residues. Furthermore, the TCEPL strategy was exploited for protein C-terminal labeling with amino reagents bearing a variety of functional groups, demonstrating its versatility and generality.

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Protein Ligation and Labeling Enabled by a C‐Terminal Tetracysteine Tag

Mo

¹

,

Lin

²

,

Chen

³

et al. 2022

Self Cite

View full text Add to dashboard Cite

The hydrazinolysis of S-cyanylated peptide provides an alternative way to afford protein αhydrazide, a key reagent used in native chemical ligation (NCL), without the aid of any inteins or enzymes. The currently used non-selective S-cyanylation, however, allows no other cysteine in the protein besides the one at the cleavage site. Herein, we report a regioselective Scyanylation and hydrazinolysis strategy achieved via the fusion of a tetracysteine tag to the C-terminal of the protein of interest. We term it tetracysteine enabled protein ligation (TCEPL). While highly selective, the strategy is applicable for proteins expressed as inclusion bodies, and this was showcased by the efficient semisynthesis of an iron-sulfur protein rubredoxin and the catalytic and hinge domains of matrix metalloprotease-14 (MMP-14) containing 207 amino acid residues. Furthermore, the TCEPL strategy was exploited for protein C-terminal labeling with amino reagents bearing a variety of functional groups, demonstrating its versatility and generality.

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Streamlined purification and characterization of Pyrococcus furiosus rubredoxins with different N-terminal modifications by reversed-phase HPLC

Cited by 4 publications

References 25 publications

Development of Nonheme {FeNO}⁷ Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Development of Nonheme {FeNO}⁷ Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Protein Ligation and Labeling Enabled by a C‐Terminal Tetracysteine Tag

Protein Ligation and Labeling Enabled by a C‐Terminal Tetracysteine Tag

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Streamlined purification and characterization of Pyrococcus furiosus rubredoxins with different N-terminal modifications by reversed-phase HPLC

Cited by 4 publications

References 25 publications

Development of Nonheme {FeNO}7 Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Development of Nonheme {FeNO}7 Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Protein Ligation and Labeling Enabled by a C‐Terminal Tetracysteine Tag

Protein Ligation and Labeling Enabled by a C‐Terminal Tetracysteine Tag

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Product

Resources

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Development of Nonheme {FeNO}⁷ Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Development of Nonheme {FeNO}⁷ Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release