SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

重原子扰动生物聚合物的光谱

基本信息

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
项目类别：
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

项目摘要

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核酸相互作用的详细信息在分子水平。这些互动是基本的在细胞代谢中的重要性以及该领域的知识可能对卫生领域的应用有很大的好处。使用将用于三胞胎状态磁性的光学检测共振（ODMR）光谱法我们将主要研究核酸结合蛋白的色氨酸残基。互动在TRP残基和核酸之间将使用 ODMR。将使用外部重原子效应来使用通过TRP残基与重原子之间的紧密相互作用 - 衍生化的核酸。另外，对激发的影响 TRP状态是由与与 ODMR将研究不细化的核酸碱基。我们建议研究结合蛋白质的络合优先对单链核酸（SSB蛋白）优先多核苷酸和寡核苷酸。一旦存在与野生型SSB的芳香堆叠相互作用已经建立，我们将研究由突变蛋白形成的复合物确定负责的特定TRP残基通过堆叠相互作用稳定复合物。地点- 选定的寡核苷酸诱变将用于形成大肠杆菌SSB和T4基因32蛋白的突变体，其基因具有由我们的合作者之一克隆。荧光和盐还将使用背部滴定来评估核酸络合物稳定性的突变。我们将使用类似于我们最近采用的策略识别大肠杆菌SSB中堆叠的TRP残基。其他突变体将研究基因32蛋白（例如琥珀突变体）可用。除了使用较重的原子衍生核酸，我们将开始使用聚（S2U）作为底物。使用 Triplet-Triplet能量转移来自该基材，我们可以产生仅在附近的那些TRP的激动状态（并与）基地。旋转对准的保护在能量传输期间将使我们能够获得有关的结构数据 TRP基堆积的复合物。除了继续我们的工作在突变的大肠杆菌SSB和T4基因32蛋白上，我们也将从丝状噬菌体PFL研究DNA结合蛋白。现在，我们将开始对真核SSB进行测量，例如 UP1和UP2来自Calf胸腺，以及Rauscher的P10蛋白鼠白血病病毒。后一种蛋白质的初步工作是完全的。