High-Resolution Protein and Nucleic Acid Footprinting

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Although X-ray crystallography and NMR spectroscopy remain the techniques of choice for obtaining high-resolution structural information on nucleic acids and nucleoprotein complexes, there is an increasing need for additional solution strategies that provide equivalent resolution, but on small quantities of material. To this end, mass spectrometry, combined with protein and nucleic acid footprinting, is emerging as a powerful tool to complement existing methodologies. The goal of the RT Biochemistry Section is to develop and implement mass spectrometric techniques to study the interaction of HIV proteins with ligands including small-molecule antagonists, other viral or cellular proteins, and finally their cognate nucleic acid substrates. High-resolution solution strategies have been applied to study the interaction of HIV-1 reverse transcriptase (RT) and integrase (IN) with a variety of ligands, including nucleic acid duplexes, small-molecule antagonists, and additional proteins [e.g., nucleocapsid (NC) protein]. Strategies successfully used with RT include site-specific attachment of photocrosslinking agents to monitor the interaction with duplex DNA and RNA/DNA, and analysis of the (-) strand initiation complex by N-hydroxysuccinamide (NHS)-biotin modification and mass spectrometric protein footprinting (Kvaratskhelia et al., PNAS, 2002). We will continue and expand these strategies to gain deeper insight into the topology of reverse transcription complexes with respect to both the protein and nucleic acid components. An extension of this aim has successfully exploited mass spectrometry to map the binding site of an IN antagonist, based on the approach of affinity acetylation (Shkriabai et al., 2004). Converting proteins into "chemical proteases" via site-specific attachment of Fe-BABE (a hydroxyl radical-generating agent) is being established as a means of studying protein/protein interactions at high resolution via mass spectroscopy. Preliminary experiments have been conducted on complexes between RT and the HIV-1 nucleocapsid protein, as well as complexes between RT and integrase.