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。此外，许多蛋白质是后修饰的。这些修饰可以调节蛋白质的活性，改变蛋白质的三级结构，或改变蛋白质的相互作用。因此，确定这些翻译后修饰是重要的。 HIV gp 120通过糖基化进行高度后修饰。我们之前已经鉴定了在特定糖基化位点观察到的聚糖类型，并注意到高甘露糖聚糖和复杂聚糖倾向于在分子表面上分离。然而，我们无法确定复合聚糖的甲硅烷基化程度。我们目前正在研究使用的碳水化合物亲和层析的组合，以浓缩糖基化的胰蛋白酶片段的gp 120，然后通过LC/MS/MS分析，以确定特定的共识糖基化位点的硅烷化的程度。我们目前还在培养表达趋化因子受体CXCR 4的细胞。我们将对分离的CXCR 4进行广泛的结构研究，并将其用于受体-配体研究。

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. Furthermore, many proteins are posttranslationally modified. These modifications can regulate the activity of the protein, alter the tertiary structure of the protein, or alter the interactions of the protein. Thus, it is important to determine these posttranslational modifications. HIV gp120 is highly posttranslationally modified by glycosylation. We have previously identified the types of glycans observed at specific glycosylation sites and have noted that high mannose glycans and complex glycans tend to be segregated on the surface of the molecule. We were not able, however, to determine the extent of silylation of the complex glycans. We are currently investigating the use of a combination of carbohydrate affinity chromatography to concentrate the glycosylated tryptic fragments of gp120 followed by LC/MS/MS analysis to determine the extent of silylation of specific consensus glycosylation sites. We are also currently growing cell cultures that express the chemokine receptor CXCR4. We will undertake extensive structural studies of the isolated CXCR4 as well as using it in our receptor-ligand studies.