Isotope-Coded Labeling for Accelerated Protein Interaction Profiling Using MS.

Protein interaction surface mapping using MS is widely applied but comparatively resource-intensive. Here, a workflow adaptation for use of isotope-coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application.

Structural and functional characterization of BaiA, an enzyme involved in secondary bile acid synthesis in human gut microbe.

Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay.

Subzero temperature chromatography for reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange mass spectrometry.

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis.

A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity.

Experimental and computational assessment of conditionally essential genes in Escherichia coli.

Genome-wide gene essentiality data sets are becoming available for Escherichia coli, but these data sets have yet to be analyzed in the context of a genome scale model. Here, we present an integrative model-driven analysis of the Keio E. coli mutant collection screened in this study on glycerol-supplemented minimal medium.

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution.

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1).

A unique RNA Fold in the RumA-RNA-cofactor ternary complex contributes to substrate selectivity and enzymatic function.

A single base (U1939) within E. coli 23S ribosomal RNA is methylated by its dedicated enzyme, RumA. The structure of RumA/RNA/S-adenosylhomocysteine uncovers the mechanism for achieving unique selectivity.

Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA: optical and EPR studies.

An unprecedented [4Fe-4S] iron-sulfur cluster was found in RumA, the enzyme that methylates U1939 in Escherichia coli 23 S ribosomal RNA (Agarwalla, S., Kealey, J. T.

Crystal structure of RumA, an iron-sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase.

RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution.

Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit.

RNA pseudouridine synthase, TruB, catalyzes pseudouridine formation at U55 in tRNA. This posttranscriptional modification is almost universally conserved and occurs in the T arm of most tRNAs. We determined the crystal structure of Escherichia coli TruB apo enzyme, as well as the structure of Thermotoga maritima TruB in complex with RNA.

Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein.

Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate. Details of the mechanism of this novel family of enzymes are unknown. Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor.

Characterization of the 23 S ribosomal RNA m5U1939 methyltransferase from Escherichia coli.

An Escherichia coli open reading frame, ygcA, was identified as a putative 23 S ribosomal RNA 5-methyluridine methyltransferase (Gustafsson, C., Reid, R., Greene, P.