High-Resolution Protein and Nucleic Acid Footprinting

虽然X射线结晶学和核磁共振光谱学仍然是获得关于核酸和核蛋白复合体的高分辨率结构信息的首选技术，但越来越需要提供同等分辨率的额外溶液策略，但只需少量材料。为此，质谱学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生物化学组的目标是开发和实施质谱学技术来研究HIV蛋白质与配体的相互作用，包括小分子拮抗剂、其他病毒或细胞蛋白质，最后是它们的同源核酸底物。 高分辨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、小分子拮抗剂和额外的蛋白质[如核衣壳(NC)蛋白]。成功应用于RT的策略包括光交联剂的定点结合以监测与双链DNA和RNA/DNA的相互作用，以及通过N-羟基丁二酰胺(NHS)-生物素修饰和质谱学蛋白质足迹分析(-)链起始复合体。我们将继续并扩展这些策略，以更深入地了解蛋白质和核酸成分方面的逆转录复合体的拓扑结构。这一目标的延伸将使用质谱学来绘制IN拮抗剂的结合部位(S)，基于“亲和乙酰化”的方法。

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. 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 will use mass spectrometry to map the binding site(s) of IN antagonists, based on the approach of "affinity acetylation."