No abstract

Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteineuracil crosslinks, which is observable as a mass increment of 151.9966 Da (C 4 H 8 S 2 O 2 ) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive Cross-linking of biomolecules combined with mass spectrometry (MS) has emerged as a powerful tool to characterize not only the tertiary and quaternary arrangements of individual biomolecules, but especially their interaction sites in biologically active complexes. By MS-based identification of the cross-linked parts or even the exact cross-linking sites of the respective biomolecules, proximity information can be derived. This has proven highly useful for computational approaches to problems such as docking or the arrangement of subunits (1-3).In principle, cross-linking can be achieved in two ways: (1) By using a chemical cross-linker that connects reactive groups of the respective biomolecules within a certain distance range, the range depending on the reagent used. (2) By generating a so-called zero-length cross-link that connects reactive groups of biomolecules that are already directly adjacent to one another. The latter is usually achieved by (UV) light-induced cross-linking, with or without the addition of compounds that induce the generation of radicals on reactive groups of the cross-linkable components or in close vicinity to them.Cross-linking in combination with MS analysis is nowadays frequently used in protein-protein interaction studies (4 -7) but can also be applied to protein-nucleic acid complexes. Indeed much attention is currently paid to their MS-based analysis owing to the crucial cellular function of many such complexes. A large variety of studies over decades have examin...

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.Methionine metabolism in mammals furnishes cells with three important metabolites, Sadenosylmethionine, needed for methylation reactions, glutathione, an important antioxidant, and taurine, an osmolyte. Homocysteine is a junction intermediate in this metabolic network and, at elevated levels, constitutes a risk factor for cardiovascular diseases, neural tube defects and Alzheimer's disease (1-3). Homocysteine concentrations are kept low by the concerted actions of at least three enzymes: betaine homocysteine methyltransferase, methionine synthase, and cystathionine beta-synthase. Impairments in the latter two enzymes and in the auxiliary protein that supports the activity of methionine synthase, i.e. methionine synthase reductase (MSR) 1 , result in inborn errors of metabolism associated with severely elevated plasma homocysteine levels (4,5).