Abstract
Herein, we document a highly optimized synthesis of the Sec-to-Cys homologue of the human selenoprotein F (SelF) through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are two one-pot manipulations, i.e. the first ligation followed by a desulfurization and the second ligation followed by the protein refolding. This way multi-milligrams of the folded synthetic protein was obtained, which set the stage for the synthesis of the natural selenoprotein. Moreover, the disulfide pairing mode of the SelF was elucidated through a combination of site-directed mutagenesis and LC-MS study. It provides not only a criterion to judge the viability of the synthetic protein, and more importantly, useful structural insights into the previously unresolved UGGT-binding domain of SelF.
Highlights
Selenoprotein F (SelF or Selenof), called the 15-kDa protein (Sep15), is a selenocysteine (Sec, U) containing eukaryotic protein localized to the endoplasmic reticulum (ER) (Gladyshev et al, 1998)
A major aim of the current work is to establish an efficient synthetic route for the Cys homolog of the mature human selenoprotein F (SelF) protein—which could later be applied for the synthesis of the Seccontaining protein
An extra C-terminal Cys residue was included in the expression plasmid for the first segment—Phe–Gly64, which was supposed to be able to transform to the corresponding thioester via a reported hydrazinolysis procedure (Adams et al, 2013; Pan et al, 2019)
Summary
Selenoprotein F (SelF or Selenof), called the 15-kDa protein (Sep15), is a selenocysteine (Sec, U) containing eukaryotic protein localized to the endoplasmic reticulum (ER) (Gladyshev et al, 1998). While lacking a typical ER retention peptide sequence, the highly conversed Cys-rich domain of SelF is believed to the key for its localization This domain is able to bind the UDP-glucose: glycoprotein glucosyltransferase (UGGT)—a large chaperone protein in the ER, it is called the UGGT binding domain (Labunskyy et al, 2005). Most of the in vitro studies of selenoproteins are carried out with the Sec-to-Cys homologue proteins In this context, the only available structure in the SelF family is reported for a fruit fly Sep, which contains no Sec residue (Scheme 1, right) (Ferguson et al, 2006), and in this case the two Cys residues (Cys and Cys82) in the so-called CXC motif forms a disulfide bond. The Cys-rich UGGT-binding domain in this structure is too flexible to be resolved in the NMR structure, as such the disulfide pairing mode in this domain is currently not known
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