Biosynthetic selenoproteins with genetically-encoded photocaged selenocysteines

Abstract

Selenocysteine is a valuable component of both natural selenoproteins and designer biocatalysts; however the availability of such proteins is hampered by technical limitations. Here we report the first general strategy for the production of selenoproteins via genetically-encoded incorporation of a synthetic photocaged selenocysteine residue in yeast cells, and provide examples of light-controlled protein dimerization and targeted covalent labeling in vitro.

Light-Mediated Sulfenic Acid Generation from Photocaged Cysteine Sulfoxide

“…In proteins, the stability of Cys sulfenic acid is determined by the surrounding microenvironment and the absence of vicinal thiols 14 and presence of basic residues 15 are often cited as key features. Protein sulfenic acid formation in vitro and in cells is most often achieved by incubation with exogenous oxidants like H 2 O 2 , organo hydroperoxides, 20 or elevating endogenous ROS production via treatment with growth factor or insulin. 10 Nevertheless, uncontrolled oxidation of reactive Cys residue(s) stemming from such methods often makes it difficult to study sulfenylation of specific proteins at defined sites within redox signaling pathways.…”

A Facile Method for Producing Selenocysteine‐Containing Proteins

“…Selenocysteine (Sec, U) is a fascinating building block for recombinant proteins: [1] it is more active and more resistant to irreversible overoxidation than cysteine (Cys), [2] is chemically modifiable, [3] and a diselenide bond is more stable than a disulfide bond in proteins. [4] Sec residues in proteins can be chemically converted into different amino acid side chains via a dehydroalanine intermediate.…”

“…[4] Sec residues in proteins can be chemically converted into different amino acid side chains via a dehydroalanine intermediate. [3a,5] Recent advances in genetic code expansion have established in vivo methods in E. coli for site-specific, UAG-dependent Sec insertion into recombinant proteins mediated by the elongation factor Tu (EF-Tu), without assistance from the Sec-dedicated elongation factor SelB and Sec-insertion sequence (SECIS) element. [6] Although wildtype tRNA Sec species have antideterminants against EF-Tu, [7] mutations in the acceptor and the T-stem of tRNA Sec remove the antideterminants.…”

Eine einfache Methode zur Produktion von Selenoproteinen

“…Selenocystein ist ein faszinierender Baustein rekombinanter Proteine: [1] Im Vergleich zu Cystein (Cys) ist Sec reaktiver, widerstandsfähiger gegen irreversible Überoxidation, [2] chemisch modifizierbar [3] und ermöglicht in Proteinen die Bildung von Diselenidbrücken, die stabiler als Disulfidbrücken sind. [4] Über Dehydroalaninintermediate können Sec-Reste darüber hinaus in Proteinen chemisch zu verschiedenen Aminosäureseitenketten umgewandelt werden.…”

“…[4] Über Dehydroalaninintermediate können Sec-Reste darüber hinaus in Proteinen chemisch zu verschiedenen Aminosäureseitenketten umgewandelt werden. [3a, 5] Die neuesten Methoden zur Erweiterung des genetischen Codes umfassen unter anderem In-vivo-Techniken zum ortsspezifischen, gerichteten, UAG-abhängigen Sec-Einbau in rekombinante Proteine unter Zuhilfenahme von Elongationsfaktor EF-Tu und sind unabhängig vom Sec-spezifischem Elongationsfaktor SelB sowie dem Sec-Insertions-Sequenz(SECIS)-Motiv. [6] Der Wildtyp von tRNA Sec schließt die Erkennung durch EF-Tu aus, [7] jedoch konnte tRNA Sec aus E. coli durch Mutationen in Akkzeptorarm und T-Stamm kompatibel zu EF-Tu gemacht werden.…”