SPECTROSCOPY OF HEAVY ATOM-PERTURBED BIOPOLYMERS

重原子扰动生物聚合物的光谱学

• 批准号: 2153172
• 负责人: AUGUST H MAKI
• 金额: $ 12.72万
• 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 1998-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2153172
• 关键词: DNA binding protein; antibiotics; chemical binding; electron spin resonance spectroscopy; fluorescent dye /probe; heavy metals; human immunodeficiency virus 1; intermolecular interaction; isomorphous substitution; linear energy transfer; methyltransferase; mutagens; nucleic acid sequence; nucleocapsid; nucleoproteins; nucleotide analog; phosphorescence; protein structure function; simian immunodeficiency virus; structural biology; thermodynamics; transcription factor; triplet state; tryptophan; virus protein

The broad long term objectives of this research project are to obtain information at the molecular level about protein-nucleic acid interaction. Of particular importance is the furthering of our understanding of factors influencing nucleic acid sequence-selectivity, and the stability of specific complexes. The interaction between proteins and nucleic acids is a fundamental component of cellular processes, as well as being essential for the assembly and functioning of infectious agents such as mammalian retroviruses. The major specific aim of the proposed research is the study of nucleic acid complexes of the nucleocapsid proteins of type 1 human immunodeficiency virus (an infectious agent leading to AIDS), HIV-1 NC p7, and of simian immunodeficiency virus, SIV NC p8. Along with all known retroviral NC proteins, p7 and p8 contain zinc finger CCHC arrays that include highly conserved aromatic residues such as trp and phe. These Zn finger arrays have been implicated in specific nucleic acid binding and packaging. Our research will focus on the use of optical detection of triplet state magnetic resonance (ODMR) of p7 and p8 complexes with heavy atom-derivatized nucleic acids to determine whether aromatic stacking interactions are involved in binding. The triplet state zero-field splitting shifts induced in trp by complex formation with a variety of nucleic acids will be used to quantitatively evaluate the contribution of aromatic stacking interactions to complex stability, and thereby, their influence on sequence selectivity. This goal will require further investigations of echinomycin (a DNA bis-intercalating antibiotic) analogs by ODMR spectroscopy as models of stacking-induced sequence selectivity. In related work, complexes of sequence-specific DNA binding proteins, E. coli trp repressor, lac repressor and Eco RI methyl transferase with 2- thiouracil- or 2-thiothymine-containing oligomers will be studied. Triplet-triplet energy transfer from the sulfur-derivatized base of DNA to protein trp residues will be utilized to obtain structural information. Extensive use will be made of mutated proteins and enzymes that involve conservative substitution of intrinsic trp residues by other amino acids as an aid in interpretation of the spectroscopic results. Any information that we can provide that bears on the origins of sequence selectivity of p7 and p8, in particular, would have important implications in such areas as the design of novel HIV antiviral agents.

该研究项目的广泛长期目标是获得 有关蛋白质-核酸相互作用的分子水平信息。 特别重要的是加深我们对因素的理解 影响核酸序列的选择性和稳定性 具体的复合物。蛋白质和核酸之间的相互作用是 细胞过程的基本组成部分，也是必不可少的 用于传染性病原体（例如哺乳动物）的组装和功能 逆转录病毒。拟议研究的主要具体目标是研究 1 型人类核衣壳蛋白的核酸复合物 免疫缺陷病毒（导致艾滋病的传染源）、HIV-1 NC p7、 和猿猴免疫缺陷病毒，SIV NC p8。连同所有已知的 逆转录病毒 NC 蛋白、p7 和 p8 含有锌指 CCHC 阵列， 包括高度保守的芳香族残基，例如trp和phe。这些锌 指状阵列与特异性核酸结合有关 包装。我们的研究将集中于利用光学检测 p7 和 p8 配合物的三重态磁共振 (ODMR) 原子衍生的核酸以确定是否芳香堆积 相互作用涉及结合。三重态零场 与多种物质形成复合物诱导的色氨酸分裂位移 核酸将用于定量评估 芳香族堆积相互作用对复合物稳定性的影响，从而， 对序列选择性的影响。这个目标还需要进一步 棘霉素（一种 DNA 双嵌入抗生素）类似物的研究 通过 ODMR 光谱作为堆积诱导的序列选择性模型。 在相关工作中，序列特异性 DNA 结合蛋白 E. 大肠杆菌 trp 阻遏物、lac 阻遏物和 Eco RI 甲基转移酶，具有 2- 将研究含硫尿嘧啶或2-硫胸腺嘧啶的低聚物。 三重态-三重态能量从 DNA 的硫衍生碱基转移到 蛋白质色氨酸残基将用于获取结构信息。 将广泛使用涉及突变的蛋白质和酶 内在色氨酸残基被其他氨基酸保守取代 帮助解释光谱结果。任何信息 我们可以提供与序列选择性的起源有关的 尤其是 p7 和 p8 将在这些领域产生重要影响 作为新型艾滋病毒抗病毒药物的设计。