STRUCTURAL STUDIES OF HIV PROTEINS

工作总结：更好地了解艾滋病毒蛋白质的结构有助于揭示蛋白质的功能并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。该项目旨在通过化学修饰、酶降解和质谱鉴定的组合来探测HIV蛋白的一级和三级结构。我们已经使用MALDI/MS和纳米电喷雾/MS/MS的组合来表征rgp 120上聚糖的异质性。该信息与截短的gp 120的已知结构一起沿着来模拟完整的，完全糖基化的GP 120。 从这个模型中，我们观察到，表面聚糖的空间分离，使高甘露糖聚集在一起，因为是复杂的glycans.We研究了gp 120与潜在的抗HIV DNA为基础的药物，使用亲和毛细管电泳与激光诱导荧光检测的相互作用。 我们发现，含有四个连续的鸟苷残基的硫代磷酸寡脱氧核苷酸结合良好，增加链长增加与gp 120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.