New Ion Mobility and Crosslinking Technologies for Analysis of Protein Complexes

描述(申请人提供)：为了执行它们的生物功能，单个蛋白质通常以一种短暂的方式结合形成复合体。对于从分子医学到物理化学的各种学科来说，了解络合物的作用方式是一个深远的科学目标。虽然X-射线衍射分析有时可以获得高细节的结构信息，但这需要有足够数量的均质材料和定义合适的结晶参数。对于大的复合体和膜蛋白来说，这两个条件通常都很难满足，因此在数据库中存放的这些结构的数量仍然相对较少。电子显微镜(EM)和小角X射线散射(SAXS)等替代方法可以确定足够尺寸的络合物的表面包络，但这些数据的解释有赖于对络合物组成的详细知识，而且一般限于均质络合物。因此，需要开发新的方法来定义具有生物医学意义的多相大分子络合物的亚单位化学计量、组成、形状和动力学，特别是在完整的生物膜和细胞器中。这一建议结合了新的交联剂策略和离子迁移率(IM)与质谱学(MS)的耦合，作为多蛋白质复合体，特别是膜复合体的高通量结构探针。基于符合MS和IM要求的小分子化学的新的交联剂策略的发展将克服蛋白质复合体分析的许多现有限制。这是朝着开发一套基于高通量质谱学的新技术迈出的第一步，这些技术将能够发现许多以前未知的多蛋白质复杂结构，并将提供用于解释结构和为蛋白质折叠计算提供约束的多肽邻近信息。重要的是，它还将为发展跟踪蛋白质复合体中相互作用动力学的方法提供基础。

DESCRIPTION (provided by applicant): To perform their biological function, individual proteins associate, often in a transient manner, to form complexes. Understanding the way complexes function is a far-reaching scientific goal for disciplines ranging from molecular medicine to physical chemistry. While high- detail structural information can sometimes be obtained by X-ray diffraction analysis, this requires the availability of a sufficient quantity of homogenous material and definition of suitable crystallization parameters. Both conditions are often difficult to meet for large complexes and for membrane proteins and thus the number of these structures deposited in databases remains relatively low. Alternative methodologies such as electron microscopy (EM) and small angle X- ray scattering (SAXS) allow determination of the surface envelope of complexes of sufficient dimensions but interpretation of these data is aided by detailed knowledge of complex composition, and is limited, in general, to homogeneous complexes. Consequently there is a need to develop new approaches that define subunit stoichiometry, composition, shape, and the dynamics of heterogeneous macromolecular complexes of biomedical importance particularly those in intact biological membranes and organelles. This proposal combines new crosslinker strategies and ion mobility (IM) coupled to mass spectrometry (MS) jointly as high-throughput structural probes for multi-protein complexes, particularly membrane complexes. The development of new crosslinker strategies based on small molecule chemistries consistent with the requirements of MS and IM will overcome many of the existing constraints for analysis of protein complexes. This is a first step towards developing a suite of new high-throughput mass spectrometry-based technologies that will enable the discovery of many previously-unknown multi-protein complex structures and will provide peptide proximity information of use for interpreting structures and providing constraints for protein folding calculations. Importantly, it will also provide a basis for developing methods for following interaction dynamics in protein complexes.