Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol (DTT) or tr...

Ribonucleotide reductases (RNRs) catalyze the formation of 2'-deoxyribonucleotides. Each polypeptide of the large subunit of eukaryotic RNRs contains two redox-active cysteine pairs, one in the active site and the other at the C-terminus. In each catalytic cycle, the active-site disulfide is reduced by the C-terminal cysteine pair, which in turn is reduced by thioredoxins or glutaredoxins. Dith...

Among all sulphhydryl oxidases involved in disulphide formation, quiescin-sulphhydryl oxidase (QSOX) is unique for its multidomain structure, protein thiol oxidation activity and highly efficient catalysis. In this study, site-directed mutagenesis and molecular modelling methods were integrated to investigate the structural and functional characteristics of QSOX, especially the importance of th...

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CADASIL is caused by more than a hundred NOTCH3 mutations. Virtually all encoded mutant proteins contain an odd number of cysteines. As such, structural changes in NOTCH3 may be the primary molecular abnormality in CADASIL. Thus, we sought evidence for structurally altered NOTCH3 protein in CADASIL tissue...

The performance of ozonation in wastewater depends on water quality and the ability to form hydroxyl radicals (·OH) to meet disinfection or contaminant transformation objectives. Since there are no on-line methods to assess ozone and ·OH exposure in wastewater, many agencies are now embracing indicator frameworks and surrogate monitoring for regulatory compliance. Two of the most promising surr...

The disulfide-based cyclic monomer, 3-methylidene-1,9-dioxa-5,12,13-trithiacyclopentadecane-2,8-dione (MTC), is statistically copolymerized with 2-hydroxypropyl methacrylate to form a range of diblock copolymer nano-objects via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(glycerol monomethacrylate) (PGMA) is employed as the hydrophilic stabilizer block in this aq...

A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, metho...

Ruthenium(II) and iridium(III) bis(terpyridine) complexes were prepared with maleimide functionalities in order to site-specifically modify yeast iso-1 cytochrome c possessing a single cysteine residue available for modification (CYS102). Single X-ray crystal structures were solved for aniline and maleimide Ru(II) 3 and Ru(II) 4, respectively, providing detailed structural detail of the complex...

Ca2+-free calmodulin was obtained by exhaustively dialyzing 2 mg protein (c = 4 mg/mL) against 1000 volumes of 2 mM EGTA, 50 mM Tris HCl and 150 mM NaCl (pH = 7.4) at 4 °C in a 0.5 mL Pierce Slide-A-LyzerTM Dialysis Cassette (3.5 K MWCO) for 2 days. As the carboxylic groups of EGTA exhibit strong IR bands in the region 1570-1630 cm-1 that overlap with the amide I' band of proteins (1600-1700 cm...

We use single-molecule force clamp spectroscopy (SMFCS) to explore the reactivity of tris(2-carboxyethyl)phosphine (TCEP), 1, 4-dl-dithiothreitol (DTT) and hydrosulfide anion (HS(-)) on disulfide bonds within a mechanically stretched polypeptide. The single-bond level bimolecular nucleophilic substitution (S(N)2) events are recorded at a series of precisely controlled temperatures so that the A...