STRUCTURAL STUDIES OF HIV PROTEINS

HIV 蛋白的结构研究

• 批准号: 6290023
• 负责人: Kenneth Tomer
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290023
• 关键词: X ray crystallography; amidation /deamidation; aspartate; chymotrypsin; computer simulation; gag protein; glutamates; human immunodeficiency virus; interleukin 4; interleukin 5; matrix assisted laser desorption ionization; microorganism culture; protein degradation; protein structure; recombinant virus; virus protein

Summary of Work: Improved understanding of the structure of HIV proteins can be useful in unraveling the function of the proteins and in understanding the mechanisms of the function of these proteins. This structural knowledge can also be useful in designing novel anti-HIV agents. X-Ray crystallography is the most accurate technique for determination of protein structures. Major drawbacks of the technique, however, are that it can be very time consuming and is dependent on the availability of protein crystals. Molecular modeling programs, on the other hand, can provide insights into the protein structure based on, amongst other things, homology with proteins whose structures are known. Although this approach is attractive, the results have often been less reliable than desired because insufficient information about the protein is available or the degree of homology with proteins of known structure is less than needed for the development of an accurate model. If information such as which amino acid residues are on the surface of the protein is available, however, the quality of the computer generated model can be significantly increased. This project is designed to probe the primary and tertiary structures of HIV proteins using a combination of chemical modifications, enzymatic degradations and mass spectrometric identification. In using commercially available sources of recombinant HIV proteins, we rapidly became aware of discrepancies between the catalog structures and the actual molecular weights of the proteins. We have developed a technique, direct analysis of affinity-bound analytes, and applied it to recombinant His-tagged proteins that are affinity bound to immobilized metal ion affinity columns to determine the actual sequence of two of these HIV proteins, rp24 and rvif. We are continuing to probe the tertiary structure of recombinant HIV proteins. Although portions of the molecule have been crystallized and the structure solved, the manner in which the two domains are configured together is not known. We are proceeding with acetylation of lysine residues on the intact native protein. Relative reactivities of the lysines have been determined. Although the relative reactivities closely match the surface accessibilities calculated from the crystal structures, there are several significant differences. Molecular modeling is being used to refine the current picture of how the two p24 domains are connected within the molecule. - Mass Spectrometry, MALDI HIV, AIDS, Protein structure, Molecular Modeling, Surface reactivites,

工作总结：对HIV蛋白质结构的进一步了解有助于揭示这些蛋白质的功能和了解这些蛋白质的功能机制。这种结构知识也可以用于设计新的抗HIV药物。X射线晶体学是测定蛋白质结构的最准确的技术。然而，该技术的主要缺点是它可能非常耗时，并且取决于蛋白质晶体的可用性。另一方面，分子建模程序可以基于与结构已知的蛋白质的同源性等来提供对蛋白质结构的见解。虽然这种方法是有吸引力的，但结果往往不如预期的可靠，因为关于蛋白质的信息不足，或者与已知结构的蛋白质的同源性程度低于开发精确模型所需的程度。然而，如果可以获得诸如蛋白质表面上的氨基酸残基等信息，则可以显着提高计算机生成模型的质量。该项目旨在使用化学修饰、酶降解和质谱鉴定的组合来探测HIV蛋白质的一级和三级结构。在使用商业上可获得的重组HIV蛋白质来源时，我们很快意识到蛋白质的目录结构和实际分子量之间的差异。我们已经开发了一种技术，直接分析的亲和力结合的分析物，并将其应用于重组组氨酸标记的蛋白质，是亲和结合到固定的金属离子亲和柱，以确定这些HIV蛋白质，rp24和rvif的实际序列。我们正在继续探索重组HIV蛋白的三级结构。虽然分子的部分已经结晶，结构也已解析，但这两个结构域结合在一起的方式尚不清楚。我们正在进行完整天然蛋白质上赖氨酸残基的乙酰化。赖氨酸的相对反应性已被确定。尽管相对反应性与晶体结构计算的表面活性非常匹配，但仍存在一些显著差异。分子模型被用来完善目前的图片如何两个p24结构域连接在分子内。- 质谱，MALDI HIV，AIDS，蛋白质结构，分子模拟，表面反应，