New Ion Mobility and Crosslinking Technologies for Analysis of Protein Complexes

用于蛋白质复合物分析的新离子淌度和交联技术

• 批准号: 8249811
• 负责人: Philip C Andrews
• 金额: $ 30.79万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8249811
• 关键词: 3-Dimensional; Affinity Chromatography; Algorithms; Architecture; Binding Proteins; Biological; Biological Process; Biology; Buffers; Cells; Cereals; Chemicals; Chemistry; Complex; Complex Mixtures; Computing Methodologies; Coupled; Crosslinker; Crystallization; Data; Data Sources; Databases; Deposition; Development; Dimensions; Discipline; Disease; Drug Delivery Systems; Drug Design; Electron Microscopy; Electrospray Ionization; Elements; Escherichia coli; Gases; Goals; Golgi Apparatus; Hybrids; Individual; Ions; Knowledge; Lead; Macromolecular Complexes; Mass Spectrum Analysis; Measurement; Membrane; Membrane Proteins; Methodology; Methods; Modeling; Molecular Machines; Molecular Medicine; Multiprotein Complexes; Organelles; Pattern; Peptides; Phase; Physical Chemistry; Property; Proteins; Rattus; Reaction; Reagent; Ribosomes; Roentgen Rays; Rough endoplasmic reticulum; Sampling; Screening procedure; Shapes; Side; Software Tools; Solutions; Solvents; Structure; Surface; System; Technology; Testing; Tissues; Work; X ray diffraction analysis; Zymogen Granules; base; chromatin remodeling; computerized tools; crosslink; ion mobility; meetings; model development; nano-electrospray; next generation; novel strategies; physical property; protein complex; protein folding; protein protein interaction; rough endoplasmic reticulum membrane; small molecule; stoichiometry; structural biology; structural genomics; surfactant

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. PUBLIC HEALTH RELEVANCE: Membrane proteins represent attractive drug targets but their unique physical properties make their structures difficult to determine and only a small fraction have had their structures determined with sufficient accuracy to be useful which limits opportunities for rational drug design. This proposal will develop high-throughput chemical and physical technologies to determine the membrane topologies of proteins and the interactions between membrane proteins that lead to function. These technologies will provide structural constraints useful in refining 3-D topology diagrams of multi-protein complexes, in de novo protein folding, defining protein-protein interactions and to study the dynamics of protein complexes in normal and diseased tissues to identify the specific protein complexes perturbed in disease.

描述（由申请人提供）：为了执行其生物学功能，单个蛋白质通常以瞬时方式缔合以形成复合物。了解复合物的功能方式是从分子医学到物理化学等学科的一个深远的科学目标。虽然有时可以通过X射线衍射分析获得高度详细的结构信息，但这需要足够量的均匀材料的可用性和合适的结晶参数的定义。对于大的复合物和膜蛋白，这两个条件通常难以满足，因此在数据库中保存的这些结构的数量仍然相对较低。替代方法如电子显微镜（EM）和小角X射线散射（SAXS）允许确定足够尺寸的复合物的表面包络，但是这些数据的解释通过复合物组成的详细知识来辅助，并且通常限于均质复合物。因此，有必要开发新的方法，定义亚基的化学计量，组成，形状，和生物医学重要性的异质大分子复合物的动力学，特别是那些在完整的生物膜和细胞器。 该建议结合了新的交联剂策略和离子迁移率（IM）耦合到质谱（MS）联合作为高通量的结构探针的多蛋白质复合物，特别是膜复合物。基于与MS和IM要求一致的小分子化学的新交联剂策略的开发将克服蛋白质复合物分析的许多现有限制。这是开发一套新的高通量质谱技术的第一步，该技术将能够发现许多以前未知的多蛋白质复合物结构，并将提供用于解释结构的肽邻近信息，并为蛋白质折叠计算提供约束。重要的是，它还将为开发蛋白质复合物中相互作用动力学的方法提供基础。 公共卫生关系：膜蛋白代表有吸引力的药物靶点，但其独特的物理性质使其结构难以确定，只有一小部分具有足够准确的结构确定是有用的，这限制了合理的药物设计的机会。该提案将开发高通量化学和物理技术，以确定蛋白质的膜拓扑结构和导致功能的膜蛋白之间的相互作用。这些技术将提供有用的结构限制，在完善的3-D拓扑图的多蛋白质复合物，从头蛋白质折叠，定义蛋白质-蛋白质相互作用，并研究蛋白质复合物在正常和患病组织的动力学，以确定特定的蛋白质复合物扰动疾病。