MASS ANALYSIS OF PROTEINS UV CROSSLINKED TO NUCLEOTIDES

描述：（改编自申请人的摘要）一般来说， 建议涉及用于研究之间相互作用的方法 蛋白质和核酸。 阐明这些相互作用是 分子生物学的中心目标，并已成为最活跃的 医学研究的追求。 拟议研究的具体目标 是开发一个通用的实验协议， 表征蛋白质的核酸结合结构域并使用 该方法用于确定核酸结合位点， 噬菌体T4基因32蛋白（gp 32），尿嘧啶-DNA糖基化酶， 大肠杆菌，并对转录终止因子rho从 大肠埃希菌 实现这些目标的实验方法将 是通过固定蛋白质和核酸之间的相互作用 紫外（UV）光诱导的光化学交联，然后 定位并鉴定特定的氨基酸和核苷酸残基 它们通过一种协议彼此共价结合， 集成了高效液相色谱（HPLC）、基质色谱（matrix- 辅助激光解吸电离（MALDI）质谱，和 电喷雾电离（ESI）质谱法。 获得的优点 通过结合这些技术应该可以大幅提高速度， 灵敏度和特异性的质谱和 色谱技术应增加的数量和类型， 生物实验，可以进行与多功能 光化学交联法 与同事合作 目前使用紫外线交联技术来研究蛋白质- 核酸相互作用将显著增加 这项研究的成功。 他们将提供一种分子生物学 这将是非常宝贵的设计，行为和 解释本提案中描述的实验，他们 将提供样本，以确保与当代相关的结果 research. 他们各自的研究将受益于回报，因为，在 在开发的每个阶段，所提出的方法将产生 否则无法获得的核酸结构信息 他们研究的结合蛋白。

DESCRIPTION: (Adapted from the applicant's abstract) 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.