STRUCTURAL STUDIES OF HIV PROTEINS

工作总结：更好地了解HIV蛋白质的结构有助于解开蛋白质的功能，并有助于了解这些蛋白质的功能机制。这种结构知识在设计新型抗艾滋病毒药物时也是有用的。X射线结晶学是确定蛋白质结构的最准确的技术。然而，这项技术的主要缺点是非常耗时，而且依赖于蛋白质晶体的可用性。另一方面，分子建模程序可以提供对蛋白质结构的洞察，其中包括与已知结构的蛋白质的同源性。尽管这种方法很有吸引力，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准确模型所需的程度。然而，如果可以获得蛋白质表面有哪些氨基酸残基之类的信息，那么计算机生成的模型的质量就可以显著提高。该项目旨在利用化学修饰、酶降解和质谱学鉴定相结合的方法来探索HIV蛋白质的一级和三级结构。在使用商业来源的重组HIV蛋白时，我们很快意识到了蛋白质的目录结构和实际分子量之间的差异。我们开发了一种技术，亲和结合分析物的直接分析，并将其应用于与固定的金属离子亲和柱亲和结合的重组组氨酸标记蛋白，以确定其中两种HIV蛋白的实际序列，rp24和rvif。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并解决了结构，但这两个结构域如何配置在一起尚不清楚。我们正在进行完整天然蛋白质上赖氨酸残基的乙酰化。测定了赖氨酸的相对反应性。尽管相对反应活性与根据晶体结构计算的表面可及性非常匹配，但仍有几个显著的差异。分子模拟被用来完善两个p24结构域如何在分子内连接的当前图景。-质谱学，MALDI艾滋病毒，艾滋病，蛋白质结构，分子模拟，表面活性，

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,