Excited state photoreaction between the indole side chain of tryptophan and halocompounds generates new fluorophores and unique modifications.

Photoreaction of indole containing compounds with chloroform and other trichlorocompounds generates products with redshifted fluorescence. In proteins, this reaction can be used for the fluorescent detection of proteins. Little characterization of products generated through the photochemical reaction of indoles with halocompounds has been done, yet is fundamental for the development of other fluorophores, protein labeling agents, and bioactive indole derivatives.

Unique Photobleaching Phenomena of the Twin-Arginine Translocase Respiratory Enzyme Chaperone DmsD.

DmsD is a chaperone of the redox enzyme maturation protein family specifically required for biogenesis of DMSO reductase in Escherichia coli. It exists in multiple folding forms, all of which are capable of binding its known substrate, the twin-arginine leader sequence of the DmsA catalytic subunit. It is important for maturation of the reductase and targeting to the cytoplasmic membrane for translocation.

Resolution-enhanced native acidic gel electrophoresis: a method for resolving, sizing, and quantifying prion protein oligomers.

The formation of β-sheet-rich prion protein (PrP(β)) oligomers from native or cellular PrP(c) is thought to be a key step in the development of prion diseases. To assist in this characterization process we have developed a rapid and remarkably high resolution gel electrophoresis technique called RENAGE (resolution-enhanced native acidic gel electrophoresis) for separating, sizing, and quantifying oligomeric PrP(β) complexes. PrP(β) oligomers formed via either urea/salt or acid conversion can be resolved by RENAGE into a clear set of oligomeric bands differing by just one subunit.

The prion protein binds thiamine.

Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier.

Stacked sets of parallel, in-register beta-strands of beta2-microglobulin in amyloid fibrils revealed by site-directed spin labeling and chemical labeling.

beta(2)-microglobulin (beta(2)m) is a 99-residue protein with an immunoglobulin fold that forms beta-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from beta(2)m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other beta(2)m aggregates.

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility.

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction.

Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry.

Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization.

Visible fluorescent detection of proteins in polyacrylamide gels without staining.

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization.

The twin-arginine leader-binding protein, DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase.

The twin-arginine translocase (Tat) pathway is involved in the targeting and translocation of fully folded proteins to the inner membrane and periplasm of bacteria. Proteins that use this pathway contain a characteristic twin-arginine signal sequence, which interacts with the receptor complex formed by the TatBC subunits. Recently, the DmsD protein was discovered, which binds to the twin-arginine signal sequences of the anaerobic respiratory enzymes dimethylsulfoxide reductase (DmsABC) and trimethylamine N-oxide (TMAO) reductase.