Incorporation of selenocysteine into proteins using peptide ligation

Abstract

Expressed protein ligation has become a frequently used technique to insert non-standard amino acids into proteins. The technique has been adapted to insert selenocysteine residues in place of cysteine residue in proteins, taking advantage of the similarity in the chemistries of sulfur and selenium. This replacement can confer unique structural and catalytic properties to enzymes and proteins. The development of this technique also allows for naturally occurring selenoproteins to be produced semisynthetically.

Figure 1

The UGA codon in the mRNA can be decoded as a sense codon if a special stem-loop structure is present in the mRNA. In prokaryotes such as E. coli, this SECIS element is located proximal to the UGA codon, while in eukaryotes, it is located in the 3′ untranslated region. There are structural differences as well, making it difficult to express a eukaryotic selenoprotein in a prokaryote such as E. coli.

Figure 2

HPLC trace of tripeptide CUG synthesized with the optimized conditions as reported by Barany [50]. Separation was achieved on a Waters C-18 column using a linear gradient of 0.1% TFA in CH₃CN and 0.1% aqueous TFA from 0% CH₃CN to 60% CH₃CN in 60 minutes. Only the L-peptide could be detected (peak 2). Some cyclized product is present (peak 1).

Figure 3

(A) Mechanism of deprotection of Sec(pMob) by TMSOTf. (B) Oxidative deprotection of Sec(pMob) peptides by I₂. (C) Possible mechanism of deprotection of a Sec(pMob) group by neighboring group participation. The Se atom can attack the adjacent disulfide resulting in the formation of a selonium ion. Removal of the pMob group is then facilitated by the presence of scavengers in the cleavage cocktail.

Figure 4

Mechanism of intein-mediated peptide ligation with peptides containing N-terminal Sec residues. The fusion protein between the protein of interest and the intein exists in equilibrium between amide and thioester forms (A–B). Thiol mediated cleavage occurs when a small molecule thiol is added at high concentration to produce a thioester-tagged protein (C). Addition of a peptide containing a N-terminal Sec residue allows for attack on the thioester to produce a selenoester (D) which rapidly rearranges to the amide form (E).

Figure 5

Ribbon drawing of RNase A showing positions of disulfide bonds and mixed selenylsulfide bonds formed with semisynthetic RNase A that incorporates selenocysteine. RNase A contains 4 disulfide bonds that occur between Cys40-Cys95, Cys84-Cys26, Cys110-Cys58, and Cys72-Cys65. Cys110 and Cys95 have been replaced with selenocysteine to create unique variants containing selenium.

Figure 6

Strategy for creating a semisynthetic thioredoxin reductase. The wild-type enzyme has a C-terminal extension consisting of the tripeptide Cys-Sec-Gly (A). A semisynthetic enzyme can be made by producing a thioester-tagged enzyme (B) missing this tripeptide and then ligating this tripeptide in vitro to create the semisynthetic enzyme (C). Such a strategy divides the protein into two distinct modules, one with selenium and one without.

Figure 7

Michaelis-Menten plot of semisynthetic and mutant thioredoxin reductases. The selenium-containing semisynthetic enzyme has very high activity compared to the mutant enzyme containing cysteine.

Scheme 1

Synthesis of the amino acid selenocystine from elemental selenium.

Scheme 2

Rate of attack on a model thioester with cysteine or selenocysteine (X=Se,S). Cystine or selenocystine were first reduced with an equilmolar amount of triscarboxyethylphosphine to produce a free thiol or selenol, respectively. The rate was then measured as a function of pH.