Improving Rate/Quality Limitations in Membrane Protein Structure Determination

改善膜蛋白结构测定中的速率/质量限制

基本信息

批准号：
7715117
负责人：
William A. Cramer
金额：
$ 65.68万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7715117
关键词：
3-Dimensional ABCG2 gene ATP phosphohydrolase ATP-Binding Cassette Transporters Abbreviations Achievement Address Advisory Committees Arginine Binding Cell Communication Cells Collaborations Color Commit Complex Coupled Crystallization Cytochrome c Reductase Cytochromes Databases Deposition Detection Detergents Development Discipline Drug Delivery Systems Early Diagnosis Electron Microscopy Ensure Environment Equipment Fluorescence Fostering G Protein-Coupled Receptor Genes G-Protein-Coupled Receptors Generations Grant Green Fluorescent Proteins Growth Home environment Institution Integral Membrane Protein Label Laboratories Lead Lipids Location Maltose Mass Spectrum Analysis Medicine Membrane Membrane Proteins Methodology Methods Micelles Molecular Molecular Sieve Chromatography Nutrient Online Systems Optics Peptide Hydrolases Phase Polymerase Chain Reaction Population Heterogeneity Postdoctoral Fellow Problem Solving Production Protein Import Proteins Proteolysis Protocols documentation Reactive Oxygen Species Research Personnel Resolution Ribose Role Scientist Screening procedure Seeds Senior Scientist Speed Structure System Techniques Testing Time Training Twin Multiple Birth Uncertainty Work base combat cytochrome b6f density drug development experience extracellular human disease improved innovation instrumentation maltose-binding protein member membrane assembly method development novel novel strategies online tutorial optical imaging programs protein complex protein structure public health relevance stigmatellin structural biology success surfactant symposium trafficking translocase two-dimensional

项目摘要

DESCRIPTION (provided by applicant): This proposal is directed to solving the problem of the slow rate of generation of high resolution structures of integral membrane proteins that can be useful as drug targets and in medicine. The group combines established expertise in membrane protein structural biology, which has led to 16 structures deposited in the Protein Data Bank, as well as cutting-edge advances in methods development. Experience has shown that the paucity of high resolution membrane protein structure is a consequence of several recognizable bottlenecks in the structure determination pipeline, including: (i) co-expression of the subunits of hetero-subunit prokaryotic proteins; (ii) expression of eukaryotic membrane proteins; (iii) determination of stability and functionality; (iv) recognition of (a) the role of lipid in the structure and in the rate of crystallization and (b) proteolytic degradation that can preclude purification of active protein. A suite of new expression approaches will be developed targeting (i) and (ii), with complementary analysis approaches in (ii). Problems (i-iv) lead to uncertainties in the formation of well-diffracting crystals, which can take weeks or months to generate. Early detection of crystal growth would greatly decrease the time required for crystal screening and allow access to a greater phase-space of crystallization conditions. We describe an innovative methodology, SONICC (second order nonlinear optical imaging of chiral crystals), for sensitive and selective detection of incipient protein crystals as small as 100 nm, prepared in screening platforms with volumes as low as 0.5 picoliter. Its ability to distinguish membrane protein crystals of the maltose transporter and cytochrome b6f complex has been confirmed. This approach will greatly speed generation of diffraction-quality 3-dimensional and 2-dimensional crystals, generated both in surfo and in meso. A range of integral membrane protein targets has been selected, with an emphasis on ABC and hetero-oligomeric proteins; (i) ABC: maltose and ribose transporters, ABCBl, ABCG2, and Sur-Kir6.2; (ii) non-ABC: twin-arginine translocase; Kdp-ATPase; NADH dehydrogenase. PUBLIC HEALTH RELEVANCE: Integral membrane proteins control all traffic between cell compartments, assembly of the membranes themselves, the supply of nutrients to the cell, its energization, and communication of the cell with the extracellular environment. The atomic structures of membrane proteins is crucial for an understanding of the molecular basis of human diseases and the development of drugs to combat them or ameliorate their consequences.

描述（由申请人提供）：该提案旨在解决可用作药物靶点和在医学中有用的完整膜蛋白高分辨率结构生成速度慢的问题。该小组结合了膜蛋白结构生物学方面的成熟专业知识（已在蛋白质数据库中存储了 16 个结构）以及方法开发方面的前沿进展。经验表明，高分辨率膜蛋白结构的缺乏是结构测定流程中几个可识别瓶颈的结果，包括：（i）异源亚基原核生物亚基的共表达蛋白质； (ii)真核膜蛋白的表达； (iii) 稳定性和功能性的确定； (iv) 认识到 (a) 脂质在结构和结晶速率中的作用以及 (b) 可能妨碍活性蛋白纯化的蛋白水解降解。将针对 (i) 和 (ii) 开发一套新的表达方法，并在 (ii) 中提供补充分析方法。问题（i-iv）导致良好衍射晶体形成的不确定性，这可能需要数周或数月才能生成。晶体生长的早期检测将大大减少晶体筛选所需的时间，并允许获得更大的结晶条件相空间。我们描述了一种创新方法 SONICC（手性晶体的二阶非线性光学成像），用于灵敏和选择性地检测小至 100 nm 的初始蛋白质晶体，这些晶体是在体积低至 0.5 皮升的筛选平台中制备的。其区分麦芽糖转运蛋白和细胞色素 b6f 复合物的膜蛋白晶体的能力已得到证实。这种方法将大大加快在表面和介观中生成的衍射质量的 3 维和 2 维晶体的生成速度。已选择一系列整合膜蛋白靶标，重点是 ABC 和异源寡聚蛋白； (i) ABC：麦芽糖和核糖转运蛋白、ABCB1、ABCG2 和苏尔基尔6.2； (ii) 非 ABC：双精氨酸转位酶； Kdp-ATP酶； NADH 脱氢酶。公共健康相关性：整合膜蛋白控制细胞区室之间的所有交通、膜本身的组装、细胞营养的供应、细胞的供能以及细胞与细胞外环境的通讯。膜蛋白的原子结构对于了解人类疾病的分子基础以及开发对抗疾病或改善其后果的药物至关重要。