The semisynthesis of nucleolar human selenoprotein H.

The human selenoprotein H is the only selenocysteine-containing protein that is located in the cell's nucleolus. In vivo studies have suggested that it plays some role in DNA binding, consumption of reactive oxygen species, and may serve as a safeguard against cancers. However, the protein has never been isolated and, as a result, not yet fully characterized. Here, we used a semi-synthetic approach to obtain the full selenoprotein H with a S43T mutation. Using biolayer interferometry, we also show that the Cys-containing mutant of selenoprotein H is capable of binding DNA with sub-micromolar affinity. Employing state-of-the-art expressed protein ligation (EPL), our devised semi-synthetic approach can be utilized for the production of numerous, hard-to-obtain proteins of biological and therapeutic relevance.

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation.

Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.

Revisiting ligation at selenomethionine: Insights into native chemical ligation at selenocysteine and homoselenocysteine.

Selenomethionine (Sem) has been incorporated recombinantly into proteins many times to elucidate their structure and function. In this paper, we revisit incorporation via chemical protein synthesis to shed light on the mechanism of native chemical ligation. The effect of chalcogen position on ligation is investigated, and selenium-containing peptide ligation is optimized. Additionally, selective methylation is performed on selenolates in a peptide in the presence of unprotected thiols.

Insights into the deselenization of selenocysteine into alanine and serine.

The development of native chemical ligation coupled with desulfurization has allowed ligation at several new ligation junctions. However, desulfurization also converts all cysteine residues in the protein sequence into alanine. Deselenization of selenocysteine, in contrast, selectively removes the selenol group to give alanine in the presence of unprotected cysteines. In this study we shed more light onto the deselenization mechanism of selenocysteine to alanine and provide optimized conditions for the reaction. The deselenization can be accomplished in one minute under anaerobic conditions to give alanine. Under aerobic conditions (oxygen saturation), selenocysteine is converted into serine.