In general, this proposal is concerned with methods used to study interactions between proteins and nucleic acids. Elucidating these interactions is one of molecular biology's central goals and has become one of the most active pursuits in medical research.
The specific aims of the proposed study are to develop a general experimental protocol for locating and characterizing the nucleic acid binding domains of proteins and to use that methodology to determine the nucleic acid binding sites on bacteriophage T4 gene 32 protein (gp32), on uracil-DNA glycosylase from Escherichia coli, and on transcription termination factor rho from Escherichia coli. The experimental approach to achieving these aims will be to fix the interaction between a protein and nucleic acid by means of ultraviolet (UV) light-induced photochemical crosslinking and then to locate and identify the particular amino acid and nucleotide residues that have been covalently bound to each other through a protocol that integrates high performance liquid chromatography (HPLC), matrix- assisted laser desorption ionization (MALDI) mass spectrometry, and electrospray-ionization (ESI) mass spectrometry. The advantages gained through combining these techniques should be substantial the speed, sensitivity, and specificity of the mass spectrometric and chromatographic techniques should increase both the number and types of biological experiments that can be conducted with the versatile photochemical crosslinking method. Collaboration with colleagues currently using UV crosslinking techniques to investigate protein- nucleic acid interactions will significantly increase the likelihood of success in this study. They will provide a molecular biological perspective that will be invaluable to the design, conduct and interpretation of the experiments described in this proposal, and they will supply samples that will assure results relevant to contemporary research. Their respective studies will benefit in return because, at every stage of development, the proposed methodology will be generating otherwise unobtainable structural information about the nucleic acid binding proteins they investigate.
| Gafken, Philip R; Doneanu, Catalin E; Bennett, Samuel E et al. (2007) Comparison of ESI-MS interfaces for the analysis of UV-crosslinked peptide-nucleic acid complexes. J Chromatogr B Analyt Technol Biomed Life Sci 860:145-52 |
| Doneanu, Catalin E; Gafken, Philip R; Bennett, Samuel E et al. (2004) Mass spectrometry of UV-cross-linked protein-nucleic acid complexes: identification of amino acid residues in the single-stranded DNA-binding domain of human replication protein A. Anal Chem 76:5667-76 |
| Doneanu, C E; Griffin, D A; Barofsky, E L et al. (2001) An exponential dilution gradient system for nanoscale liquid chromatography in combination with MALDI or nano-ESI mass spectrometry for proteolytic digests. J Am Soc Mass Spectrom 12:1205-13 |
