Mechanistic investigations of the pseudouridine synthase RluA using RNA containing 5-fluorouridine.

The pseuoduridine synthases (psi synthases) isomerize uridine (U) to pseudouridine (psi) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the psi synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The psi synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U.

Metaproteomic analysis of Chesapeake Bay microbial communities.

Background: Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results: This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes).

The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB.

All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base.

The pseudouridine synthases: revisiting a mechanism that seemed settled.

RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB.

Generating protein sequence tags by combining cone and conventional collision induced dissociation in a quadrupole time-of-flight mass spectrometer.

The goal of proteomics research is to be able to identify and quantify the vast numbers of proteins within an organism or tissue. "Top-down" methods address this goal without the need for proteolytic digestion prior to mass analysis. We report here an approach for top-down protein identification that has been implemented on a commercially available, unmodified Qq-TOF mass spectrometer.

Formation of oligomers in secondary organic aerosol.

The formation of oligomeric molecules, an important step in secondary organic aerosol production, is reported. Aerosols were produced by the reaction of alpha-pinene and ozone in the presence of acid seed aerosol and characterized by exact mass measurements and tandem mass spectrometry. Oligomeric products between 200 and 900 u were detected with both electrospray ionization and matrix-assisted laser desorption ionization.

Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures.

The field of proteomics requires methods that offer high sensitivity and wide dynamic range. One of the strategies used to improve the dynamic range is sample prefractionation, such as microsolution isoelectric focusing (IEF). We have modified a commercial solution IEF instrument, the Rotofor, to prefractionate protein mixtures by carrier ampholyte-free solution IEF.