Integrated Methods for Structural Elucidation of Proteins and Macromolecular Comp

蛋白质和大分子化合物结构解析的综合方法

• 批准号: 8297329
• 负责人: Evan R Williams
• 金额: $ 27.02万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8297329
• 关键词: 26S proteasome; Acids; Affect; Alzheimer' s Disease; Amides; Amino Acids; Anthrax disease; Apoptosis; Area; Back; Biological; Biological Process; Blood capillaries; California; Cattle; Cell physiology; Charge; Chemistry; Collaborations; Complex; Complex Mixtures; Creutzfeldt-Jakob Syndrome; Crystallography; Cystic Fibrosis; Deuterium; Development; Disease; Disease Progression; Dissociation; Electron Microscopy; Electron Transport; Electrons; Electrospray Ionization; Enzymes; Gases; Goals; Heating; Higher Order Chromatin Structure; Hydrogen; Immune response; Individual; Infection; Ions; Kinetics; Lead; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methodology; Methods; Molecular; Molecular Models; Multiprotein Complexes; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Play; Post-Translational Protein Processing; Prion Diseases; Proteasome Inhibitor; Protein translocation; Proteins; Reagent; Relative (related person); Research; Resolution; Role; Screening procedure; Signal Transduction; Solutions; Spectrometry; Speed; Structure; Structure-Activity Relationship; Therapeutic Agents; Time; Transcriptional Regulation; Travel; Tube; Universities; Vertebral column; anthrax toxin; cancer therapy; capillary; improved; instrument; interest; ion mobility; macromolecular assembly; mass spectrometer; meter; molecular assembly/self assembly; molecular modeling; molecular size; multicatalytic endopeptidase complex; novel; pressure; protein aggregation; protein complex; protein function; protein misfolding; protein structure; small molecule; stoichiometry; tandem mass spectrometry; tool

DESCRIPTION (provided by applicant): Most cellular functions are performed by large molecular assemblies and information about the structures and functions of these higher-order complexes can lead to an improved understanding of many biological processes, including how diseases progress, at the molecular level. The goal of the proposed research is to develop an integrated approach to determining detailed information about the higher-order structures of proteins and macromolecular assemblies that combines high-resolution ion mobility spectrometry (IMS) with solution-phase chemistry and hydrogen deuterium exchange (H/DX) with supercharging tandem mass spectrometry, and apply these methods to determine detailed information about the structures and structural transitions that occur in important macromolecular complexes where only limited information has been obtained using conventional high resolution structural methods. A low-pressure drift tube apparatus will be constructed and interfaced to a QTOF mass spectrometer to obtain absolute collision cross sections of large macromolecular complexes. These cross sections can provide information about structure/function relationships of the complex, as well as provide reference measurements for others using commercially available traveling wave IMS instruments that only provide relative cross section measurements. This IMS capability will be used to develop new H/DX methods that use supercharging reagents to unfold, dissociate, and highly charge the constituent molecules in the ms or sub-ms time frame of ion formation by electrospray, which eliminates any back exchange that can occur with conventional acid quenching methods. The goal is to obtain backbone amide exchange rates with close to individual amino acid resolution using electron transfer or electron capture dissociation tandem mass spectrometry, which have been shown to minimize gas-phase H/D scrambling and the concomitant loss of exchange rate information. The effect of increased charging obtained with these reagents on any gas-phase H/D scrambling that may occur will be investigated. A proteolytic 'nicking' strategy will be pursued to significantly increase the molecular size range where this top-down method can be used. A novel method to measure assembly kinetics at short times will be developed and used to find molecular frameworks that have the potential to be developed into effective drugs for treatment of anthrax infection and proteasome inhibitors in development for cancer therapy. These integrated approaches can potentially increase the speed, sensitivity, and molecular size range for which structural information can be obtained with high fidelity, and will provide complementary information to other structural methods, including electron microscopy, NMR, crystallography, and molecular modeling. These approaches will be applied to investigate the structures and structural transitions that occur in several complexes where limited information has been obtained, including Anthrax toxin complexes, and the 26S proteasome. PUBLIC HEALTH RELEVANCE: This research is aimed at developing a fast and sensitive integrated approach for obtaining detailed information about the structure/function relationships of large molecular assemblies. This methodology can be used where conventional structural methods provide only limited information, and should be applicable to a wide range of areas of biomedical interest, including discovery of therapeutic agents aimed at regulating biomolecular complex formation, signal transduction, immune response, transcriptional regulation, protein translocation, apoptosis, and protein misfolding and aggregation that play a role in many diseases, including Alzheimer's disease, cystic fibrosis, spongiform encephalopathies (e.g., Mad Cow or Creutzfeldt Jakob disease), and even some cancers.

描述（由申请人提供）：大多数细胞功能是由大分子组装体执行的，有关这些高级复合物的结构和功能的信息可以提高对许多生物过程的理解，包括在分子水平上疾病如何进展。拟议研究的目标是开发一种综合方法来确定有关蛋白质和大分子组装体的高阶结构的详细信息，该方法将高分辨率离子迁移谱（IMS）与溶液相化学和氢氘交换（H / DX）与增压串联质谱法，并应用这些方法来确定有关重要大分子复合物中发生的结构和结构转变的详细信息，而使用传统的高分辨率结构方法只能获得有限的信息。将构建低压漂移管装置并将其与 QTOF 质谱仪连接，以获得大分子复合物的绝对碰撞截面。这些横截面可以提供有关复合体结构/功能关系的信息，并为其他使用仅提供相对横截面测量的市售行波 IMS 仪器的其他人提供参考测量。这种 IMS 功能将用于开发新的 H/DX 方法，该方法使用增压试剂通过电喷雾在离子形成的毫秒或亚毫秒时间范围内对组成分子进行展开、解离和高度充电，从而消除了任何反向交换，发生在传统的酸淬火方法中。目标是使用电子转移或电子捕获解离串联质谱法获得具有接近单个氨基酸分辨率的主链酰胺交换率，这已被证明可以最大限度地减少气相 H/D 扰乱和随之而来的交换率信息的损失。将研究使用这些试剂获得的增加的电荷对可能发生的任何气相 H/D 扰乱的影响。将采用蛋白水解“切口”策略来显着增加可以使用这种自上而下方法的分子大小范围。将开发一种在短时间内测量组装动力学的新方法，并用于寻找有潜力开发成治疗炭疽感染的有效药物和正在开发的用于癌症治疗的蛋白酶体抑制剂的分子框架。这些集成方法有可能提高获得高保真度结构信息的速度、灵敏度和分子尺寸范围，并将为其他结构方法（包括电子显微镜、核磁共振、晶体学和分子建模）提供补充信息。这些方法将用于研究在获得有限信息的几种复合物中发生的结构和结构转变，包括炭疽毒素复合物和 26S 蛋白酶体。 公共健康相关性：本研究旨在开发一种快速、灵敏的综合方法，以获得有关大分子组装体的结构/功能关系的详细信息。该方法可用于传统结构方法仅提供有限信息的情况，并且应适用于广泛的生物医学领域，包括发现旨在调节生物分子复合物形成、信号转导、免疫反应、转录调节、蛋白质易位、细胞凋亡以及蛋白质错误折叠和聚集在许多疾病中发挥作用，包括阿尔茨海默病、囊性纤维化、海绵状脑病（例如疯牛病或克雅氏病）病），甚至某些癌症。