STRUCTURAL STUDIES OF HIV PROTEINS

工作总结：提高对HIV蛋白结构的理解有助于揭示蛋白质的功能和理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最精确的技术。然而，该技术的主要缺点是它可能非常耗时，并且依赖于蛋白质晶体的可用性。另一方面，分子建模程序可以根据与已知结构的蛋白质的同源性，提供对蛋白质结构的深入了解。虽然这种方法很有吸引力，但结果往往不像期望的那样可靠，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准确模型所需的程度。然而，如果诸如蛋白质表面的氨基酸残基等信息是可用的，则可以显著提高计算机生成模型的质量。该项目旨在利用化学修饰、酶解和质谱鉴定相结合的方法探测HIV蛋白的一级和三级结构。在使用商业上可获得的重组HIV蛋白来源时，我们很快意识到目录结构与蛋白质实际分子量之间的差异。我们开发了一种直接分析亲和结合分析物的技术，并将其应用于与固定金属离子亲和柱亲和结合的重组his标记蛋白，以确定其中两种HIV蛋白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，结构也已解出，但这两个结构域的组合方式尚不清楚。我们正在对完整的天然蛋白上的赖氨酸残基进行乙酰化。测定了赖氨酸的相对反应活性。虽然相对反应性与晶体结构计算的表面可及性非常接近，但也存在一些显著的差异。分子模型被用来完善两个p24结构域如何在分子内连接的当前图像。-质谱，MALDI， HIV，艾滋病，蛋白质结构，分子建模，表面反应性，

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,