Selenoproteins containing the 21st amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low p Ka, high reactivity, and redox property of Sec also afford unique routes to protein modification and engineering. However, natural Sec incorporation requires idiosyncratic translational machineries that are dedicated to Sec and species-dependent, which makes it challenging to recombinantly prepare selenoproteins with high Sec specificity. As a consequence, the function of half of human selenoproteins remains unclear, and Sec-based protein manipulation has been greatly hampered. Here we report a new general method enabling the site-specific incorporation of Sec into proteins in E. coli. An orthogonal tRNAPyl-ASecRS was evolved to specifically incorporate Se-allyl selenocysteine (ASec) in response to the amber codon, and the incorporated ASec was converted to Sec in high efficiency through palladium-mediated cleavage under mild conditions compatible with proteins and cells. This approach completely obviates the natural Sec-dedicated factors, thus allowing various selenoproteins, regardless of Sec position and species source, to be prepared with high Sec specificity and enzyme activity, as shown by the preparation of human thioredoxin and glutathione peroxidase 1. Sec-selective labeling in the presence of Cys was also demonstrated on the surface of live E. coli cells. The tRNAPyl-ASecRS pair was further used in mammalian cells to incorporate ASec, which was converted into Sec by palladium catalyst in cellulo. This robust and versatile method should greatly facilitate the study of diverse natural selenoproteins and the engineering of proteins in general via site-specific introduction of Sec.fications.

The versatile chemistry of the genetically encoded amino acid selenocysteine (Sec) is employed in Nature to expand the reactivity of enzymes. In addition to, its role in biology, Sec is used in protein engineering to modify folding, stability, and reactivity of proteins, to introduce conjugations and to facilitate reactions. However, due to limitations related to Sec’s insertion mechanism in Nature, much of the production of Sec containing peptides and proteins relies on synthesis and semisynthesis. Here, we review recent advances that have enabled the assembly of complicated selenoproteins, including novel uses of protecting groups for solid phase peptide synthesis, rapid selenoester driven chemical ligations and versatile expressed protein ligations.

Fe oxidation is one of Earths major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our ability to track the contributions of Fe-oxidizing microbes is limited by our relatively incomplete knowledge of microbial Fe oxidation mechanisms, particularly in neutrophilic Fe-oxidizers. The genomes of many Fe-oxidizers encode homologs to an outer-membrane cytochrome (Cyc2) that has been shown to oxidize Fe in two acidophiles. Here, we demonstrate the Fe oxidase function of a heterologously expressed Cyc2 homolog derived from a neutrophilic Fe oxidizer. Phylogenetic analyses show that Cyc2 from neutrophiles cluster together, suggesting a common function. Sequence analysis and modeling reveal the entire Cyc2 family is defined by a unique structure, a fused cytochrome-porin, consistent with Fe oxidation on the outer membrane, preventing internal Fe oxide encrustation. Metatranscriptomes from Fe-oxidizing environments show exceptionally high expression of cyc2, supporting its environmental role in Fe oxidation. Together, these results provide evidence that cyc2 encodes Fe oxidases in diverse Fe-oxidizers and therefore can be used to recognize microbial Fe oxidation. The presence of cyc2 in 897 genomes suggests that microbial Fe oxidation may be a widespread metabolism.

Selenoprotein K (SELENOK) is a selenocysteine (Sec)-containing protein localized in the endoplasmic reticulum (ER) membrane where it interacts with the DHHC6 (where single letter symbols represent Asp-His-His-Cys amino acids) enzyme to promote protein acyl transferase (PAT) reactions. PAT reactions involve the DHHC enzymatic capture of palmitate via a thioester bond to cysteine (Cys) residues that form an unstable palmitoyl-DHHC intermediate, followed by transfer of palmitate to Cys residues of target proteins. How SELENOK facilitates this reaction has not been determined. Splenocyte microsomal preparations from wild-type mice versus SELENOK knockout mice were used to establish PAT assays and showed decreased PAT activity (~50%) under conditions of SELENOK deficiency. Using recombinant, soluble versions of DHHC6 along with SELENOK containing Sec92, Cys92, or alanine (Ala92), we evaluated the stability of the acyl-DHHC6 intermediate and its capacity to transfer the palmitate residue to Cys residues on target peptides. Versions of SELENOK containing either Ala or Cys residues in place of Sec were equivalently less effective than Sec at stabilizing the acyl-DHHC6 intermediate or promoting PAT activity. These data suggest that Sec92 in SELENOK serves to stabilize the palmitoyl-DHHC6 intermediate by reducing hydrolyzation of the thioester bond until transfer of the palmitoyl group to the Cys residue on the target protein can occur.

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization