No abstract

Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. In order to fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved 11 CXX 14 C active site were chemically synthesized. Grx3, Grx3(C11U) and Grx3(C14U) exhibited redox potentials of −194, −260 and −275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (−309 mV) and thioredoxin (Trx) (−270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a 'central atom' in the exchange reaction. The 10 2 to 10 4 -fold increase in the rate of Trx reduction by the seleno-Grx3 analogs demonstrates that Oxidoreductases containing either selenenylsulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts.

The deselenization of selenocysteine selectively removes the selenol group to give alanine under anaerobic conditions or serine under aerobic conditions (oxygen saturation).

Selenocysteine, the selenium-containing analogue of cysteine, is the twenty-first proteinogenic amino acid. Since its discovery almost fifty years ago, it has been exploited in unnatural systems even more often than in natural systems. Selenocysteine chemistry has attracted the attention of many chemists in the field of chemical biology owing to its high reactivity and resulting potential for various applications such as chemical modification, chemical protein (semi)synthesis, and protein folding, to name a few. In this Minireview, we will focus on the chemistry of selenium and selenocysteine and their utility in protein chemistry.

Targeted insertion of a non‐native diselenide cross‐link into a cysteine‐rich protein can be exploited to direct the early stages of oxidative folding so as to avoid accumulation of unproductive intermediates that limit folding efficiency. This simple strategy could facilitate the production of many difficult‐to‐fold peptides and proteins.

The synthesis and kinetic parameters of a comprehensive set of 4-OT analogues with arginine (X = NH2+) to citrulline (X = O) substitutions at positions 11, 39, and 61 are reported. These data suggest that the main contribution of Arg39' ' to catalysis is by electrostatic stabilization of the anionic transition state leading to intermediate 2, and not by hydrogen bonding.

Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125-residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C-terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N-terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analogue in which the active-site histidine was substituted with the unnatural and isosteric amino acid β-thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.

Tip-enhanced Raman spectroscopy is a surface sensitive analytical method that combines the advantages of scanning probe microscopy and Raman spectroscopy. It holds great promises for imaging of biological samples with high spatial resolution (10−50 nm), well below the optical diffraction limit. It offers the opportunity to directly localize and identify proteins and their conformation in a complex (e.g., native) environment. Tip-enhanced Raman (TER) spectra in the socalled "gap-mode" configuration with a metal tip in scanning tunnelling feedback with a metal substrate coated with different proteins (bovine serum albumin, immunoglobulin G, trypsin, and β-lactoglobulin) as well as of model octapeptides (with and without an aromatic amino acid residue) are presented. The goal was to determine if it is possible to reliably assign marker bands for proteins and if different secondary structures of proteins can be distinguished in their gap-mode TER spectra as reliably as by IR and conventional Raman spectroscopy. It is shown that contrary to the presented conventional Raman spectra of proteins the amide I mode, which is widely used to identify secondary structure motifs of proteins, is not visible in gap-mode TERS. Aromatic modes are prominent and can be used as reliable marker bands for imaging of proteins in a complex environment.