High-Resolution Protein and Nucleic Acid Footprinting

虽然X射线晶体学和NMR光谱学仍然是获得核酸和核蛋白复合物的高分辨率结构信息的首选技术，但越来越需要提供等效分辨率的额外解决方案策略，但需要少量材料。为此，质谱法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生物化学部分的目标是开发和实施质谱技术，以研究HIV蛋白与配体（包括小分子拮抗剂）、其他病毒或细胞蛋白以及最终其同源核酸底物的相互作用。 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）与各种配体的相互作用，所述配体包括核酸双链体、小分子拮抗剂和另外的蛋白质[例如，核衣壳（NC）蛋白]。成功使用RT的策略包括位点特异性连接的光交联剂，以监测与双链DNA和RNA/DNA的相互作用，并通过N-羟基琥珀酰胺（NHS）-生物素修饰和质谱蛋白质足迹分析（-）链起始复合物。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相对于蛋白质和核酸组分的拓扑结构。这一目标的延伸将基于“亲和乙酰化”方法，使用质谱法来绘制IN拮抗剂的结合位点。"

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."