Harnessing selenocysteine reactivity for oxidative protein folding

Metanis, Norman; Hilvert, Donald

doi:10.1039/c4sc02379j

Cited by 66 publications

(71 citation statements)

References 36 publications

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“…Combined with mechanistic insights, these results suggested that COCs could cross the lipid bilayer membrane as molecular walkers through successive thiol–dichalcogenide exchange reactions along disulfide tracks in membrane proteins (Figure A) after reacting with proximal exofacial thiols (Figure B) . Such processes could cause temporary local protein denaturation and transient micellar defects in the membrane to allow the passage of large substrates. The transferrin receptor mentioned above is a possible candidate of a disulfide track, having intermolecular disulfide bridges at the entry into the membrane (C89, C98) and nearby thiols, although possibly acylated (C62, C67) .…”

Section: Figurementioning

confidence: 94%

Probing for Thiol‐Mediated Uptake into Bacteria

et al. 2019

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Cellular uptake mediated by cyclic oligochalcogenides (COCs) is emerging as a conceptually innovative method to penetrate mammalian cells. Their mode of action is based on dynamic covalent oligochalcogenide exchange with cellular thiols. To test thiol‐mediated uptake in bacteria, five antibiotics have been equipped with up to three different COCs: One diselenolane and two dithiolanes. We found that the COCs do not activate antibiotics in Gram‐negative bacteria. In Gram‐positive bacteria, the COCs inactivate antibiotics that act in the cytoplasm and reduce the activity of antibiotics that act on the cell surface. These results indicate that thiol‐mediated uptake operates in neither of the membranes of bacteria. COCs are likely to exchange with thiols on the inner, maybe also on the outer membrane, but do not move on. Concerning mammalian cells, the absence of a COC‐mediated uptake into bacteria observed in this study disfavors trivial mechanisms, such as passive diffusion, and supports the existence of more sophisticated, so far poorly understood uptake pathways.

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Section: Figurementioning

confidence: 94%

Probing for Thiol‐Mediated Uptake into Bacteria

et al. 2019

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“…Furthermore, because diselenide bonds are more stable than disulfides (relative to their respective reduced forms), substituting Sec residues in strategic positions can improve folding efficiency even via non‐native pairing followed by protein structure‐driven rearrangement . This counterintuitive outcome was demonstrated in the case of the bovine pancreatic trypsin inhibitor (BPTI), a classical model of protein folding: pairwise replacement of Cys5 and Cys14 with Sec yielded a non‐native diselenide bond that was associated with enhanced folding efficiency because trapped intermediates that otherwise confound the folding of WT‐BPTI were sidestepped …”

Section: Introductionmentioning

confidence: 99%

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin

Weil-Ktorza

Rege

Lansky

et al. 2019

Chemistry A European J

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Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, CysA6 and CysA11 (which form the internal intrachain A6–A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid‐phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild‐type B chain‐di‐S‐sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long‐standing challenge in chemical insulin synthesis. The affinity of the Se‐insulin analogue for the lectin‐purified insulin receptor was indistinguishable from that of WT‐insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔGu ≈0.8 kcal mol−1). In accordance with such enhanced stability, reductive unfolding of the Se‐insulin analogue and resistance to enzymatic cleavage by Glu‐C protease occurred four times more slowly than that of WT‐insulin. 2D‐NMR and X‐ray crystallographic studies demonstrated a native‐like three‐dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.

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“…The oxidative folding of reduced wild type (WT)‐BPTI and MT‐BPTI was studied in parallel under various conditions. We first tested under standard conditions, wherein the reduced WT‐BPTI folded to form two one‐disulfide intermediates [30–51] and [5–55]. As the reaction proceeded, the two‐disulfide intermediates N′ and N* start to appear; these intermediates are stable over time because of their quasi‐native structures .…”

Section: The Case Of Bptimentioning

confidence: 99%

Miklós Bodanszky Award Lecture: Selective chalcogen chemistry to study protein science

Metanis

Dardashti

Mousa

et al. 2019

Journal of Peptide Science

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In recent decades, chemical protein synthesis and the development of chemoselective reactions—including ligation reactions—have led to significant breakthroughs in protein science. Among them are a better understanding of protein structure‐function relationships, the study of protein posttranslational modifications, exploration of protein design, unnatural amino acid incorporation, and the study of therapeutic proteins and protein folding. Chalcogen chemistry, especially that of sulfur and selenium, is quite rich, and we have witnessed continuous progress in this field in recent years. In this short review, we will instead summarize three stories that we have recently presented on chalcogen chemistry and its impact on protein science, which was presented in the Miklós Bodanszky Award Lecture at the 35th European Peptide Society Meeting in Dublin, Ireland, 26 August 2018.

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Harnessing selenocysteine reactivity for oxidative protein folding

Abstract: Turbo-charged folding with selenium: targeted replacement of cysteines in proteins with selenocysteines is a valuable strategy for increasing the rates of oxidative protein folding, altering folding mechanisms, and rescuing kinetically trapped intermediates.

Cited by 66 publications

References 36 publications

Probing for Thiol‐Mediated Uptake into Bacteria

Probing for Thiol‐Mediated Uptake into Bacteria

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin

Miklós Bodanszky Award Lecture: Selective chalcogen chemistry to study protein science

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