Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd^III−Gd^III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd^III were indistinguishable from Gd^III−Gd^III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.     

Deliberately Losing Control of C−H Activation Processes in the Design of Small-Molecule-Fragment Arrays Targeting Peroxisomal Metabolism

Combined photochemical arylation, “nuisance effect” (S_NAr) reaction sequences have been employed in the design of small arrays for immediate deployment in medium-throughput X-ray protein–ligand structure determination. Reactions were deliberately allowed to run “out of control” in terms of selectivity; for example the ortho-arylation of 2-phenylpyridine gave five products resulting from mono- and bisarylations combined with S_NAr processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.     

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).     

A magnetic resonance imaging study of planum temporale asymmetry in men with developmental dyslexia

The influence of ionic strength and composition on the binding and inhibition of human leukocyte elastase by glycosaminoglycans with variable degree and position of sulfation was investigated. The kinetic mechanism of inhibition had a hyperbolic, mixed-type character with a competitive component that was promoted by low ionic strength, reduced by phosphate ions, and which also depended on the substrate and glycosaminoglycan structure. Enzyme binding was a cooperative phenomenon that varied with ionic strength and composition. The inhibition patterns correlated with the cationic character of elastase and with the distribution of arginines on its molecular surface, most notably with residues located in the vicinity of the substrate binding region. The order of affinity for elastase binding was chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate, iduronate-containing derivatives being superior with respect to the glucuronate-containing counterparts. Additional sulfation at both the 4- and 6- positions or at the N- and 4-positions of the N-acetylgalactosamine moiety decidedly improved the inhibitory efficiency. The results highlight a fundamental physiological role of enzyme-glycosaminoglycan interactions. In the azurophil granule of the human polymorphonuclear neutrophil, elastase and other enzymes are bound to a matrix of chondroitin 4-sulfate because this is the only glycosaminoglycan that simultaneously offers good binding for enzyme compartmentalization together with prompt release from the bound state at the onset of phagocytosis.