Identification of Subunit Interfaces in Protein Complex

蛋白质复合物中亚基界面的鉴定

• 批准号: 6571774
• 负责人: Peter E. Prevelige
• 金额: $ 20.58万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6571774
• 关键词: SDS polyacrylamide gel electrophoresis; affinity labeling; biotin; capsid; chemical synthesis; crosslink; cysteine; human immunodeficiency virus 1; lysine; mass spectrometry; protein protein interaction; protein purification; protein structure; proteomics; technology /technique development; virion

DESCRIPTION (provided by applicant): The objective of this proposal is to develop a general methodology for the rapid identification of intersubunit interfaces in protein complexes, and utilize it to identify the subunit/subunit interfaces in immature and mature HIV-1 capsids, The strategy will be to synthesize a family of biotin-tagged chemical cross-linkers with both specific and non-specific cross-linking activity and use them as distance constraints to position subunits of known atomic structure (in this case the structural proteins of HIV) relative to one another in space, To achieve the necessary structural resolution the cross-linked amino acid residue pairs will be identified by mass spectrometry. This project extends the parent grant (AI44626) whose mission is to utilize hydrogen/deuterium exchange studies to identify intersubunit interfaces and dynamic motions in HIV capsids. The novel component of this proposal is the synthesis of biotin tagged cross-linkers, which can be rapidly purified using strepavidin affinity techniques coupled with mass spectrometry for identification of the cross-linked peptides. Such cross-linkers are not commercially available. Our preliminary data indicates that while the cross-linking data can be used to pack subunits, its widespread applicability is limited by the inherent difficulty of detecting a small number of cross-linked peptides against a background of a large number of uncross-linked peptides. Biotin tagged cross-linkers can be rapidly purified from the complex digests and analyzed by mass spectrometry resulting in increases in sensitivity and throughput. As a result the technique will become a generally applicable tool for merging data from the structural genomics and proteomics initiatives.

描述(由申请人提供)：该建议的目标是开发一种通用的方法来快速识别蛋白质复合体中的亚基间界面，并利用它来识别未成熟和成熟的HIV-1衣壳中的亚基/亚单位界面，该策略将是合成一族具有特定和非特定交联活性的生物素标记的化学交联剂，并将它们用作距离约束，以定位已知原子结构的亚基(在这种情况下是HIV的结构蛋白质)在空间中相对于另一个亚基，为了实现必要的结构分辨率，将通过质谱学鉴定交联的氨基酸残基对。该项目扩展了父母赠款(AI44626)，其任务是利用氢/氚交换研究来确定艾滋病毒衣壳中亚单位间的界面和动态运动。该方案的新颖之处在于合成了生物素标记的交联剂，利用链霉亲和素亲和技术结合质谱仪鉴定交联肽，可以快速纯化交联肽。这种交联剂在商业上是买不到的。我们的初步数据表明，虽然交联数据可以用来包装亚基，但其广泛的适用性受到在大量未交联肽的背景下检测少量交联肽的固有困难的限制。生物素标记的交联剂可以从复杂的消化中快速纯化并进行质谱分析，从而提高了灵敏度和吞吐量。因此，该技术将成为一种普遍适用的工具，用于合并来自结构基因组学和蛋白质组学倡议的数据。