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. Here, we demonstrate that DmsD exhibits an irreversible photobleaching phenomenon upon 280 nm excitation irradiation. The phenomenon is due to quenching of the tryptophan residues in DmsD and is dependent on its folding and conformation. We also show that a tryptophan residue involved in DmsA signal peptide binding (W87) is important for photobleaching of DmsD. Mutation of W87, or binding of the DmsA twin-arginine signal peptide to DmsD in the pocket that includes W72, W80, and W91 significantly affects the degree of photobleaching. This study highlights the advantage of a photobleaching phenomenon to study protein folding and conformation changes within a protein that was once considered unusable in fluorescence spectroscopy.

Redox enzyme maturation proteins (REMPs) are system-specific chaperones required for the maturation of complex iron sulfur molybdoenzymes that are important for anaerobic respiration in bacteria. Although they perform similar biological roles, REMPs are strikingly different in terms of sequence, structure, systems biology, and type of terminal electron acceptor that it supports for growth. Here we critically dissect current knowledge pertaining to REMPs of the nitrate reductase delta superfamily, specifically recognized in Escherichia coli to include NarJ, NarW, TorD, DmsD, and YcdY, also referred to as the NarJ REMP subfamily. We show that NarJ subfamily members share sequence homology and similar structural features as revealed by alignments performed on structurally characterized REMPs. We include an updated phylogenetic analysis of subfamily members, justifying their classification in this subfamily. The structural and functional roles of each member are presented herein and these discussions suggest that although NarJ subfamily members are related in sequence and structure, each member demonstrates remarkable uniqueness, validating the concept of system-specific chaperones.

BackgroundThe twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence.Methodology/Principal FindingsThe aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species.Conclusions/SignificanceTatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host.

Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000-g supernatant of optic nerves was analyzed for incorporation of 3H-amino acids into proteins. No incorporation of 3H-amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S-200 column (removing molecules less than 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4-5 times for Lys and 2-13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5-15 times more [3H]Lys and [3H]Leu into proteins than did the shafts.(ABSTRACT TRUNCATED AT 250 WORDS)

A 150,000-g supernatant from axoplasm of the giant axon of the stellate nerve of the squid and from rat sciatic and goldfish optic nerves was found to be able to incorporate covalently [3H]putrescine and [3H]spermidine into an exogenous protein (N,N'-dimethylcasein). Incorporation of radioactivity was inhibited by CuSO4, a specific inhibitor of transglutaminases, the enzymes mediating these reactions in other tissues. Analysis of pH and temperature range and enzyme kinetics displayed characteristics predicted for transglutaminase-mediated reactions. Transglutaminase activity increased during regeneration of both vertebrate nerves, but greater activity was found in segments of nerve containing no intact axons than in either intact segments or in segments containing regenerating axons. Polyacrylamide gel electrophoresis of endogenous modified proteins (in the absence of N,N'-dimethylcasein) showed labeling of 18-, 46- and 200-kilodalton proteins by both [3H]putrescine and [3H]spermidine. Analysis of the protein-bound radioactivity from intact and regenerating rat sciatic nerves demonstrated it to be predominantly in the form of the parent radioactive polyamine. These experiments demonstrate the covalent modification of proteins by polyamines at low levels in squid axoplasm and at relatively higher levels in rat sciatic and goldfish optic nerves. In the latter two cases, the activity of these modification reactions may be due in part to the modification of axonal proteins, but the majority of the activity occurs in nonneuronal cells of the nerve.