High-Resolution Protein and Nucleic Acid Footprinting

尽管x射线晶体学和核磁共振波谱学仍然是获得核酸和核蛋白复合物高分辨率结构信息的首选技术，但对提供同等分辨率的附加溶液策略的需求越来越大，但仅限于少量材料。为此，质谱与蛋白质和核酸足迹相结合，正在成为补充现有方法的有力工具。RT生物化学部门的目标是开发和实施质谱技术，以研究HIV蛋白与配体的相互作用，包括小分子拮抗剂、其他病毒或细胞蛋白，以及它们的同源核酸底物。高分辨率溶液策略已被应用于研究HIV-1逆转录酶（RT）和整合酶（IN）与多种配体的相互作用，包括核酸双链、小分子拮抗剂和其他蛋白质（如核衣壳（NC）蛋白）。RT成功使用的策略包括光交联剂的位点特异性附着，以监测与双链DNA和RNA/DNA的相互作用，以及通过n -羟基丁二胺(NHS)-生物素修饰和质谱蛋白足迹分析（-）链起始复合物（Kvaratskhelia等人，PNAS, 2002）。我们将继续并扩展这些策略，以更深入地了解逆转录复合物的拓扑结构，包括蛋白质和核酸成分。基于亲和乙酰化的方法，这一目标的延伸已经成功地利用质谱法绘制了IN拮抗剂的结合位点（Shkriabai et al., 2004）。通过Fe-BABE（一种羟基自由基生成剂）的位点特异性附着，将蛋白质转化为“化学蛋白酶”，这是一种通过质谱以高分辨率研究蛋白质/蛋白质相互作用的方法。RT与HIV-1核衣壳蛋白之间的复合物以及RT与整合酶之间的复合物已经进行了初步实验。

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.