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。我们利用MALDI/MS和纳米电喷雾/MS/MS相结合的方法表征了rgp120上聚糖的异质性。该信息与已知的截断gp120的结构一起用于模拟完整的、完全糖基化的gp120的结构。从这个模型中，我们观察到表面聚糖在空间上是分离的，因此高甘露糖和复杂的聚糖聚集在一起。我们利用激光诱导荧光检测亲和毛细管电泳技术研究了gp120与潜在的抗hiv DNA药物的相互作用。我们发现含有四个相邻鸟苷残基的硫酸寡脱氧核苷酸结合良好，并且增加的链长增加了与gp120的结合。

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.We have characterized the heterogeneity of the glycans on rgp120 using a combination of MALDI/MS and nanoelectrospray/MS/MS. This information was used along with the known structure of a truncated gp120 to model a structure of the intact, fully glycosylated gp120. From this model we observed that the surface glycans are spatially segregated so that high mannose are clustered together as are the complex glycans.We have investigated the interactions of gp120 with potential anti-HIV DNA based drugs using affinity capillary electrophoresis with laser induced fluorescence detection. We found that phosphorothioate oligodeoxynucleotides containing four contiguous guanosine residues bind well and that increased chain length increases binding with gp120.