Cell‐Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

Abstract

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd III À Gd III distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd III were indistinguishable from Gd III À Gd III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.

Paramagnetic Chemical Probes for Studying Biological Macromolecules

“…An emerging solution may be offered by the site-specific incorporation of photocaged selenocysteine, which, following decaging by UV irradiation, delivers a site that is much more reactive toward alkylating probes than cysteine thiol groups. 727,728 The potential of harnessing this strategy for attaching double-armed probes is clear but has not yet been explored. Also the incorporation of an unnatural amino acid containing a stable nitroxide radical has not yet been achieved in a way that would dethrone the MTSL tag as the most popular spin label, partly because nitroxides are sensitive to reduction in live cells and partly because no aminoacyl-tRNA synthetase is available that is specific for a nitroxide amino acid featuring a short and rigid linker between nitroxide group and protein backbone.…”

“…A generally satisfying solution has not yet been found, however, because either the resulting linker to the target protein is quite long (as in the case of, for example, probes attached by click chemistry), the unnatural amino acid disrupts the protein structure (as in the case of, for example, phosphoserine), or the amino acid causes protein precipitation upon metal binding (as in the case of the bipyridyl and 8-hydroxy-quinoline amino acids). An emerging solution may be offered by the site-specific incorporation of photocaged selenocysteine, which, following decaging by UV irradiation, delivers a site that is much more reactive toward alkylating probes than cysteine thiol groups. , The potential of harnessing this strategy for attaching double-armed probes is clear but has not yet been explored. Also the incorporation of an unnatural amino acid containing a stable nitroxide radical has not yet been achieved in a way that would dethrone the MTSL tag as the most popular spin label, partly because nitroxides are sensitive to reduction in live cells and partly because no aminoacyl-tRNA synthetase is available that is specific for a nitroxide amino acid featuring a short and rigid linker between nitroxide group and protein backbone.…”

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time‐Resolved Luminescence Studies

“…The selenol group of selenocysteine (Sec) is much more nucleophilic than the thiol group of cysteine and recently developed technology for genetic encoding of a photocaged selenocysteine residue, in principle, enables site‐specific installation of a selenocysteine residue, although the protein yields obtainable are still too low for routine use. [76] As solvent‐exposed selenocysteine residues are highly prone to forming Se−Se bonds, free selenol groups can be maintained only in the presence of reducing agents, [77] which are incompatible with tags that contain activated disulfide bonds. In the present work, we therefore tested the compatibility of the C12 tag with reducing agents and its potential for ligation to selenocysteine.…”

Chemoselective Copper-Mediated Modification of Selenocysteines in Peptides and Proteins

“…Furthermore, we demonstrated the high efficiency of this strategy in the preparation of cyclic peptides and the modification of larger and more complex peptides and proteins (ubiquitin and SDF1) at micromolar concentrations. Although all tested peptides and proteins were prepared by chemical synthesis, this methodology can be applied to selenoproteins that are prepared using expression systems. − This new approach is complementary to the reported umpolung method by Cohen et al, , as our radical reaction is highly chemoselective, does not require ligands for copper ions, works in neat aqueous solutions on peptides and proteins, and tolerates all functional groups. Further work to explore the labeling and fine-tuning of more complex biological molecules in vivo and in vitro with this technology is ongoing in our laboratory.…”