SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

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 对细胞新陈代谢的重要性，以及这一领域的知识可能 对卫生领域的应用大有裨益，并促进其应用。 将使用三重态磁的光学检测 共振(ODMR)光谱，我们将主要用它来研究 核酸结合蛋白的色氨酸残基。互动 色氨酸残基和核酸之间的关系将使用 ODMR。将利用外部重原子效应 通过色氨酸残基与重原子之间的紧密相互作用- 衍生的核酸。此外，对兴奋的影响 与色氨酸堆积相互作用产生的色氨酸状态 未衍生的核酸碱基将由ODMR进行研究。我们 建议研究结合蛋白的络合作用 优先于单链核酸(SSB蛋白) 多核苷酸和寡核苷酸。一旦人类的存在 与野生型SSB的芳香族堆积作用已被 ，我们将研究突变蛋白质形成的复合体 确定特定的色氨酸残留物，这些残留物对 通过堆积作用稳定络合物。站点- 将采用选择性寡核苷酸突变来形成 大肠杆菌SSB和T4基因32蛋白的突变体，其基因有 是由我们的一个合作者克隆的。荧光和盐- 此外，还将使用反向滴定来评估 突变对核酸复合体稳定性的影响。我们会 使用与我们最近采用的策略类似的策略 鉴定大肠杆菌SSB中堆积的色氨酸残留物。其他突变体 例如琥珀突变体之类的基因32蛋白，将在他们 都是可用的。除了使用重原子衍生的核 酸，我们将开始使用聚(S2U)作为底物。vbl.使用 三胞胎-三胞胎能量从这个衬底转移，我们可以生产 只有那些色氨酸在其附近的激发态。 (并与基地堆叠在一起)。自旋排列守恒 在能量转移过程中将使我们能够获得关于 色氨酸碱基堆积复合体。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 对丝状噬菌体Pf1的DNA结合蛋白进行了研究。 我们现在将开始对真核生物的表面活性物质进行测量，例如 来自小牛胸腺的UP1和UP2以及来自Rauscher的p10蛋白 小鼠白血病病毒。关于后一种蛋白质的初步工作是 完成。

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.