FT-ICR MS studies of RNA-protein/RNA-ligand interactions

Interactions between ribonucleic acids (RNA) and proteins or other ligands such as metabolites are pervasive and key to many fundamental biological processes, including protein biosynthesis and infection by RNA viruses. For a thorough understanding of these important interactions, RNA-protein and RNA-ligand complexes are commonly investigated by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, both of which require relatively large quantities of sample material. Moreover, "isotopic labeling" of samples for NMR spectroscopy can be expensive, and X-ray crystallography can become impossible if the complex fails to crystallize properly. From our recent work on proteins, data from the literature, and preliminary studies for the proposed project, we found convincing evidence that in the gas phase environment of a mass spectrometer, electrostatic interactions between binding partners can be stronger than their covalent bonds. Coincidentally, electrostatic interactions are common elements of RNA- protein binding, and can frequently be found in RNA-ligand complexes. In this project, we will investigate in detail these unusually strong interactions, and put them to use in the development of a new mass spectrometry (MS) approach for the detection of RNA-protein and RNA-ligand complexes and the characterization of their binding interfaces. The proposed MS approach will complement NMR spectroscopy, X-ray crystallography, and other biophysical or biochemical methods, and offers distinct advantages, among them high sensitivity, and at the same time provides extensive sequence information of the binding partners.

The aim of the research project "FT-ICR MS studies of RNA-protein/RNA-ligand interactions" was to gain a fundamental understanding of the electrostatic interactions between ribonucleic acids (RNA) and proteins or other ligands in the gas phase, thus providing a solid basis for the development of top-down native electrospray ionization (ESI) mass spectrometry (MS) methodology for the detection of RNA-protein complexes and the characterization of binding interfaces. For this purpose, we studied (1) the stability of native RNA-protein and RNA-ligand complexes in the gas phase, (2) the competition between RNA backbone cleavage and noncovalent bond dissociation at different activation energies, and (3) RNA binding of small molecules as models for functional groups, moieties, or segments of proteins. We found that peptide or ligand dissociation in the gas phase was generally preceded by intermolecular proton transfer from the peptide or ligand to the RNA, a process favored at very high (high proton affinity of RNA anions) or very low (high number of available H+) net negative charge of the complexes. At intermediate net charge (0.2 - 0.4 charges/nt), the interactions between ligand and RNA are preserved during CAD provided that their number is sufficiently high, which is typically the case for peptides and ligands such as aminoglycosides. Moreover, we determined the experimental window providing full sequence coverage from c and y fragments while preserving the intermolecular interactions regarding net charge and activation energy. On the basis of these results, we were able to develop top-down native ESI MS for the detection of RNA-protein and RNA-ligand complexes and the characterization of binding interfaces by low energy collisionally activated dissociation, and to use this approach in studies of transactivating (tat) peptide binding to transactivation responsive (TAR) RNA and rev peptide binding to rev response element (RRE) RNA from human immunodeficiency virus type 1 (HIV-1) as well as aminoglycoside binding to a riboswitch aptamer.