SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

重原子扰动生物聚合物的光谱

• 批准号: 3249975
• 负责人: AUGUST H MAKI
• 金额: $ 12.06万
• 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/3249975
• 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.

该研究计划的长期目标是获得 有关蛋白质-核酸相互作用的详细信息 在分子水平上。 这些相互作用具有基础性 在细胞代谢中的重要性，并且该领域的知识可能 对健康领域大有裨益，并促进其应用。 将利用光学检测三重态磁 共振（ODMR）光谱，我们将主要研究 核酸结合蛋白的色氨酸残基。 互动 Trp 残基和核酸之间的差异将使用以下方法进行评估 ODMR。 利用外部重原子感应效应 通过色氨酸残基和重原子之间的密切相互作用 衍生化的核酸。 此外，对兴奋的影响 Trp 的状态是由与 未衍生化的核酸碱基将通过 ODMR 进行研究。 我们 提议研究结合的蛋白质的络合 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 的芳香堆积相互作用已被证实 建立后，我们将研究突变蛋白形成的复合物 识别负责的特定色氨酸残基 通过堆积相互作用稳定复合物。 地点- 将采用选定的寡核苷酸诱变来形成 大肠杆菌SSB和T4基因32蛋白的突变体，其基因具有 被我们的一位合作者克隆了。 荧光和盐- 也将使用反滴定来评估以下效果 突变对核酸复合物稳定性的影响。 我们将 使用与我们最近采用的策略类似的策略 识别大肠杆菌 SSB 中堆积的色氨酸残基。 其他突变体 基因 32 蛋白，如琥珀突变体，将被研究，因为它们 可用。 除了使用重原子衍生的核酸外 酸，我们将开始使用聚（s2U）作为基材。 使用 从该基底的三重态-三重态能量转移，我们可以产生 仅那些位于附近的色氨酸的激发态 （并与底座堆叠在一起）。 自旋排列守恒 在能量转移过程中，我们能够获得结构数据 色氨酸碱基堆叠复合物。 除了继续我们的工作 关于突变型大肠杆菌 SSB 和 T4 基因 32 蛋白，我们还将 研究来自丝状噬菌体 Pfl 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 UP1 和 UP2，以及来自 Rauscher 的 p10 蛋白 鼠白血病病毒。 对后一种蛋白质的初步工作是 完全的。