Structural Studies Of Hiv Proteins

HIV 蛋白质的结构研究

• 批准号: 6673003
• 负责人: Kenneth Tomer
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6673003
• 关键词: HIV envelope protein gp120; X ray crystallography; affinity chromatography; computer simulation; cytokine receptors; electrospray ionization mass spectrometry; glycosylation; human immunodeficiency virus; liquid chromatography mass spectrometry; matrix assisted laser desorption ionization; microorganism culture; protein degradation; protein structure function; recombinant virus

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.

工作摘要：对HIV蛋白结构的理解的提高可用于揭示蛋白质的功能以及理解这些蛋白质功能的机制。这种结构知识也可用于设计新型的抗HIV药物。 X射线晶体学是确定蛋白质结构的最准确技术。但是，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，分子建模程序可以根据已知结构的蛋白质提供对蛋白质结构的见解。尽管这种方法很有吸引力，但结果通常比所需的可靠性不那么可靠，因为有关该蛋白质的信息不足，或者与已知结构蛋白质的同源性程度少于开发准确模型所需的蛋白质。此外，许多蛋白质经过翻译后修饰。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很重要。 HIV GP120通过糖基化进行了高度的翻译后修饰。我们先前已经鉴定出在特定的糖基化位点观察到的聚糖类型，并注意到高甘露糖的聚糖和复杂的聚糖倾向于在分子表面隔离。但是，我们无法确定复合物聚糖的硅烷基水平。我们目前正在研究使用碳水化合物亲和力色谱的组合来浓缩GP120的糖基化胰蛋白酶片段，然后进行LC/MS/MS分析，以确定特定共识糖基化位点的硅化程度。我们目前还在生长表达趋化因子受体CXCR4的细胞培养物。我们将对分离的CXCR4进行广泛的结构研究，并在我们的受体配体研究中使用它。