Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode

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.

Chemical synthesis of human selenoprotein F and elucidation of its thiol-disulfide oxidoreductase activity

“…In analogy to the synthesis of the SelF(U65C) analogue, 7 the full-length protein was disconnected into three segments at Gly41–Cys42 and Gln74–Ala75. Noted that the Gln74 was mutated to an Ala residue in order to obtain a stable peptide hydrazide segment, and according to previous experience 7 this mutation should not affect the protein folding and function ( vide infra ). Unlike most of the reported synthetic Sec-containing proteins where the Sec residue is placed in the N-terminus of the peptide, i.e.…”

“…S52†), which agrees well the reported NMR structure of the fruit fly Sep15 protein as well as the SelF(U65C) homologue (both with a disulfide bond instead). 4,7…”

“… 4 In this context, the disulfide pairing mode of SelF(U65C) has been elucidated in our previous work through site-directed mutagenesis and enzymatic digestion (see Fig. 1B ), 7 the clear evidence of a selenenylsulfide bond in the CGU motif is, however, still lacking. Notably, a few examples have been reported using the genetic code expansion technology to incorporate Sec site-specifically.…”

A shelf stable Fmoc hydrazine resin for the synthesis of peptide hydrazides

“…[17] This method avoids the need for strong oxidizing conditions, enabling broader compatibility with common synthetic peptide functionalities, and has been widely adopted in a broad range of applications. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] The increased use of C-terminal α-hydrazides in peptide chemistry creates a demand for robust synthetic tools for accessing these moieties. A number of methods have been published to allow access to C-terminal hydrazides on both synthetic [15,16,38] and expressed [15,39,40] peptides and proteins.…”

A Shelf Stable Fmoc Hydrazine Resin for the Synthesis of Peptide Hydrazides

“…17 This method avoids the need for strong oxidizing conditions, enabling broader compatibility with common synthetic peptide functionalities, and has been widely adopted in a broad range of applications. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] The increased use of C-terminal α-hydrazides in peptide chemistry creates a demand for robust synthetic tools for accessing these moieties. A number of methods have been published to allow access to C-terminal hydrazides on both synthetic 15,16,38 and expressed 15,39,40 peptides and proteins.…”