SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

重原子扰动生物聚合物的光谱

• 批准号: 3249973
• 负责人: AUGUST H MAKI
• 金额: $ 11.4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-01-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3249973
• 关键词: DNA; DNA binding protein; Escherichia coli; RNA; Rauscher's virus; bacterial proteins; bacterial virus; binding proteins; chemical binding; chemical structure function; fluorescence; heavy metals; intermolecular interaction; metal complex; microwave spectrometry; molecular biology; molecular energy level; molecular stacking; mutant; nuclear magnetic resonance spectroscopy; oligonucleotides; phosphorescence; point mutation; polymers; polynucleotides; protein sequence; site directed mutagenesis; triplet state; tryptophan; virus protein

The long-term objective of this research program is to acquire detailed information regarding protein-nucleic acid interactions at a molecular level. These interactions are of fundamental importance in cellular metabolism, and knowledge in this area may be of great benefit to, and foster applications in the health area. Use will be made of optical detection of triplet state magnetic resonance (ODMR) spectroscopy with which we will study mainly the tryptophan residues of nucleic acid-binding proteins. Interactions between Trp residues and nucleic acids will be evaluated using ODMR. Use will be made of the external heavy atom effect induced by close interactions between Trp residues and heavy atom- derivatized nucleic acids. In addition, effects on the excited states of Trp which result from stacking interactions with underivatized nucleic acid bases will be investigated by ODMR. We propose to study the complexing of proteins which bind preferentially to single-stranded nucleic acids (SSB proteins) with polynucleotides and oligonucleotides. Once the existence of aromatic stacking interactions with wild type SSBs has been established, we will study the complexes formed by mutant proteins to identify specific Trp residues which are responsible for stabilization of the complex through stacking interactions. Site- selected oligonucleotide mutagenesis will be employed to form mutants of E. coli SSB and of T4 gene 32 protein, whose genes have been cloned by one of our collaborators. Fluorescence and salt- back titrations will be used, as well, to evaluate the effects of mutations on the stability of the nucleic acid complexes. We will use a strategy similar to that which we have employed recently to identify the stacked Trp residues in E. coli SSB. Other mutants of gene 32 protein such as an amber mutant, will be studied as they are available. In addition to using heavy atom-derivatized nucleic acids, we will begin to employ poly (s2U) as a substrate. Using triplet-triplet energy transfer from this substrate, we can produce the excited states of only those Trp which are in the vicinity of (and stacked with) the bases. Conservation of spin alignment during energy transfer will enable us to obtain structural data on the Trp-base stacked complex. In addition to continuing our work on mutant E. coli SSBs and T4 gene 32 proteins, we also will investigate the DNA binding protein_from the filamentous phage Pfl. We will now begin to make measurements on eukaryotic SSBs, such as UP1 and UP2 from calf thymus, and the p10 protein from Rauscher murine leukemia virus. Preliminary work on the latter protein is complete.

这项研究计划的长期目标是获得 关于蛋白质-核酸相互作用的详细信息 在分子水平上。 这些相互作用是基本的 细胞代谢的重要性，以及该领域的知识， 对健康领域有很大的益处，并促进其在健康领域的应用。 将使用三重态磁性的光学检测。 共振（ODMR）光谱，我们将主要研究 核酸结合蛋白的色氨酸残基。 相互作用 Trp残基和核酸之间的关系将使用 ODMR。 将利用外部重原子效应诱导 通过色氨酸残基和重原子之间的密切相互作用， 衍生的核酸。 此外，对兴奋的 Trp的状态是由与 将通过ODMR研究未衍生化的核酸碱基。 我们 建议研究蛋白质的复合， 优先于单链核酸（SSB蛋白）， 多核苷酸和寡核苷酸。 一旦存在 与野生型SSB的芳族堆积相互作用已经被 建立，我们将研究突变蛋白质形成的复合物 以确定特定的色氨酸残基， 通过堆叠相互作用稳定复合物。 研究中心- 选择的寡核苷酸诱变将用于形成 突变株E. coli SSB和T4基因32蛋白，其基因具有 被我们的一个合作者克隆了 荧光和盐- 还将使用反滴定法评价以下因素的影响： 突变对核酸复合物的稳定性的影响。 我们将 使用类似于我们最近使用的策略， 鉴定E. coli SSB. 其他突变体 基因32蛋白，如琥珀突变体，将被研究，因为他们 都是可用的。 除了使用重原子衍生的核酸外， 酸，我们将开始采用聚（s2 U）作为衬底。 使用 三重态-三重态能量转移，我们可以产生 只有那些在附近的色氨酸的激发态 (and与）基地堆叠。 自旋排列守恒 将使我们能够获得结构数据， Trp-碱基堆积复合物。 除了继续我们的工作 关于突变体E. coli SSBs和T4基因32蛋白，我们还将 研究丝状噬菌体Pf 1的DNA结合蛋白。 我们现在将开始对真核SSB进行测量，例如 来自小牛胸腺的UP 1和UP 2，以及来自Rauscher的p10蛋白 鼠白血病病毒 后一种蛋白的初步工作是 完成.