Identification of Subunit Interfaces in Protein Complex

描述（申请人提供）：本研究的目的是建立一种快速鉴定蛋白质复合物中亚基间界面的通用方法，并利用该方法鉴定未成熟和成熟HIV-1衣壳中的亚基/亚基界面，该策略将是合成一个家族的生物素标记的化学交联剂，具有特异性和非特异性的交叉，连接活性，并使用它们作为距离约束，以在空间中相对于彼此定位已知原子结构的亚基（在这种情况下为HIV的结构蛋白）。为了实现必要的结构分辨率，将通过质谱法鉴定交联的氨基酸残基对。该项目扩展了母基金（AI 44626），其使命是利用氢/氘交换研究来识别HIV衣壳中的亚基间界面和动态运动。该提案的新组成部分是生物素标记的交联剂的合成，其可以使用链霉亲和素亲和技术结合质谱法快速纯化以鉴定交联肽。此类交联剂不可商购。我们的初步数据表明，虽然交联数据可用于包装亚基，但其广泛适用性受到固有困难的限制，即在大量未交联肽的背景下检测少量交联肽。生物素标记的交联剂可以从复合物中快速纯化，并通过质谱法进行分析，从而提高灵敏度和通量。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。

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.