MASS ANALYSIS OF PROTEINS UV CROSSLINKED TO NUCLEOTIDES

描述：(改编自申请人的摘要)总的来说，这 提案涉及用于研究相互作用的方法 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 从事医学研究。拟议研究的具体目标 是开发一种通用的实验方案来定位和 鉴定蛋白质的核酸结合域并使用 确定其上的核酸结合位点的方法 噬菌体T4基因32蛋白(Gp32)在尿嘧啶-DNA糖基酶上的表达 大肠杆菌和转录终止因子Rho的表达 大肠埃希菌。实现这些目标的试验性方法将 通过以下方式固定蛋白质和核酸之间的相互作用 紫外光诱导的光化学交联 定位和鉴定特定的氨基酸和核苷酸残基 它们已经通过一种协议相互共价绑定，该协议 集高效液相色谱(HPLC)、基质 辅助激光解吸电离(MALDI)质谱仪 电喷雾电离(ESI)质谱仪。获得的优势 通过结合这些技术应该有实质性的速度， 质谱学的灵敏度和特异度 层析技术应该增加细菌的数量和种类 生物实验可以用多功能的 光化学交联法。与同事协作 目前使用紫外光交联技术来研究蛋白质- 核酸的相互作用将显著增加 在这项研究中取得成功。他们将提供一种分子生物学 这将对设计、实施和 对本提案中描述的实验的解释，以及它们 将提供样本以确保与当代相关的结果 研究。他们各自的学习将得到回报，因为，在 在开发的每个阶段，建议的方法都会产生 否则就无法获得有关核酸的结构信息 他们研究的是结合蛋白。

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.