KEY INTERACTIONS FOR HIV 1 RT STABILITY AND DIMERIZATION

HIV 1 RT 稳定性和二聚化的关键相互作用

• 批准号: 6686359
• 负责人: Clyde A. Hutchison
• 金额: $ 29.1万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-15 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6686359
• 关键词: RNA directed DNA polymerase; chemical models; computer simulation; crosslink; dimer; enzyme activity; enzyme inhibitors; high performance liquid chromatography; human immunodeficiency virus 1; hydrogen bond; microorganism culture; protein folding; protein protein interaction; protein purification; site directed mutagenesis; transfection /expression vector; tryptophan; virus protein

Description: The primary focus of this proposal is the identification of amino acid interactions that are critical for the formation of stable HIV-1 reverse transcriptase (RT) heterodimers. A detailed understanding of these interactions will potentially provide a basis for the rational design of new classes of therapeutically useful inhibitors for the processes involved in forming an active HIV-1 RT. The proposed experiments will distinguish between interactions critical for 1) protein folding, 2) stability of the folded protein structure, and 3) formation of the p66/p51 heterodimer. Approaches to the identification of peptide inhibitors for various stages in the synthesis and maturation of the RT heterodimer will also be investigated. The experimental approach will rely on efficient methods for production and screening of specific amino acid replacement mutations. The pro-pol-int coding sequence of HIV-1 is expressed in E. coli where it is processed by the encoded protease to produce p66/p51 heterodimer that is indistinguishable from that produced in the infected human cell. Mutant design will be guided by the 3-dimensional X-ray structure of the RT, and by previous mutational analysis which has identified amino acid residues critical for folding and/or stability. To identify interactions critical for dimerization, we have developed a simple assay for stable heterodimers which can be performed on crude extracts of E. coil expressing the RI. The C-terminus of the p66 subunit is tagged with six His residues so that it can be retained on nickel spin columns. The bound RT is eluted, analyzed by Western blotting, and visualized with an anti-RI monoclonal antibody. The wild-type RT shows both p66 and also p51 which is retained on the column in a stable dimer with the His tagged p66. Mutants that destabilize the dimer show little or no bound p51. Mutants at critical residues that produce reduced amounts of correctly folded active RT will be studied to determine whether the residue is critical only during the folding process, or for stability of the correctly folded product. It is expected that residues involved in important steps in the folding process can be identified in this way. Computer modeling of the structures of related RTs (HIV-2, SIV and others) will be performed to study the degree of evolutionary conservation of interactions critical for folding, stability, and dimerization of RT. These RTs will be cloned in our expression system to allow direct study of conserved critical interactions.

描述：这项提案的主要焦点是氨基的鉴定 对形成稳定的HIV-1逆转至关重要的酸相互作用 转录酶(RT)异二聚体。对这些互动的详细了解 可能会为合理设计新的 在治疗上有用的抑制剂，参与形成的过程 活动性HIV-1RT。拟议的实验将区分相互作用 对1)蛋白质折叠，2)折叠蛋白质结构的稳定性至关重要， 3)p66/p51杂二聚体的形成。辨别身份的方法 在合成和成熟的不同阶段的多肽抑制物 RT异源二聚体也将被研究。 实验方法将依赖于有效的生产方法和 特定氨基酸替换突变的筛选。PROPOL-INT编码 HIV-1的序列在大肠杆菌中表达，并由编码的 产生与之没有区别的p66/p51杂二聚体的蛋白酶 在受感染的人类细胞中产生。突变体的设计将由 RT的三维X射线结构，并经前期突变分析 它已经确定了对折叠和/或稳定性至关重要的氨基酸残基。 为了确定对二聚化至关重要的相互作用，我们开发了一个简单的 可对E. 表示RI的线圈。P66亚基的C末端标记有六个 他的残基可以保留在镍的旋转柱上。界限RT为 洗脱，用Western blotting分析，并用抗RI的单抗显影 抗体。野生型RT既显示了p66，也显示了保留在 在带有组氨酸标记的p66的稳定二聚体中。破坏人类社会稳定的突变体 二聚体显示很少或没有结合的p51。 关键残基上的突变体产生的正确折叠量减少 将对活性RT进行研究，以确定残留物是否只是关键的 在折叠过程中，或为了正确折叠的产品的稳定性。 预计残基参与了折叠过程中的重要步骤 可以用这种方式来识别。相关结构的计算机模拟 将进行RTS(HIV-2、SIV和其他)以研究 相互作用的进化守恒对折叠、稳定性和 RT的二聚化。这些RT将被克隆到我们的表达系统中，以允许 直接研究保守的关键相互作用。