Sulfur And Selenium In Peptides And Proteins: Part I – Chemoselective Methods For Disulfide Bond Formation, Part Ii – Function Of Selenium In Enzymes.
Selenocysteine (Sec), the 21st proteogenic amino acid, was first identified in 1976 by Thressa Stadtman. In proteins, Sec replaces the far more common sulfur-containing amino acid cysteine (Cys). A key question since Stadtman’s discovery is: Why does Sec replace Cys? This question is especially relevant since Cys-orthologs of Sec-enzymes catalyze the identical reaction with only slightly reduced efficiency, and incorporation of Sec into a protein is much more complicated and bioenergetically costly compared to Cys. The study of selenoproteins is very difficult because Sec is incorporated into proteins by recoding a UGA stop codon as a sense codon. Production of recombinant selenoproteins involves reconstituting the recoding machinery. Alternatively, selenoproteins can be produced using a combination of recombinant DNA technology and peptide synthesis. While the development of solid phase peptide synthesis has provided a synthetic route for studying Sec, the Sec side chain requires the use of sturdy protecting groups (PGs) during synthesis.
Work herein first addresses complications associated with Sec and Cys PGs, which until now have required harsh conditions for removal. My work has developed facile new methods for the deprotection of Sec and Cys residues. For Sec, we found that the use of DTNP to remove various PGs with subsequent ascorbolysis results in a Sec-selenol. Likewise, we developed 2,2’-dipyridyl diselenide (PySeSePy) to deprotect Cys, which can be used with subsequent ascorbolysis to provide a Cys-thiol. Notably, we found the ascorbolysis step to be chemoselective; ascorbate can reduce a selenosulfide bond, but not a formed disulfide bond. We harnessed this chemoselectivity for the synthesis of peptides that contain multiple disulfide bonds, which we demonstrate by synthesizing guanylin and tachyplesin-1 using PySeSePy as a chemical tool.
Another chemical tool that we utilize to explore selenoprotein chemistry is alpha-methyl selenocysteine (αMe)Sec. This unique amino acid has a methyl group in place of its α-H. We found that a peptide containing (αMe)Sec (compared to a Sec-peptide control), showed enhanced stability when incubated in oxygenated buffer for prolonged periods of time. We also utilized our (αMe)Sec-peptide as a glutathione peroxidase mimic to reduce peroxides, and postulate that this peptide could serve as a therapeutic in times of high oxidative stress.
Finally, it is now commonly accepted in the field that selenoproteins evolved to resist oxidative stress. Herein, we expand this hypothesis: Sec replaces Cys in proteins to resist all types of electrophilic stress. We found that when Sec residues are alkylated by reactive biological electrophiles (such as acrolein), the formed adduct can be reversed. There are many potential mechanisms of reversal, but we provide evidence supporting a selenoxide elimination mechanism using a mutant form of thioredoxin reductase that contains (αMe)Sec in place of the native Sec residue at the C-terminal active site. Taken together, the works described in this dissertation expand the chemical toolbox for the study of Se-containing biomolecules and provides new hypotheses for the chemical role of Se in proteins.