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

• 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
• 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

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）：该建议旨在解决可以用作药物靶标和医学的积分膜蛋白高分辨率结构的生成速度缓慢的问题。该小组结合了膜蛋白结构生物学方面的既定专业知识，该专业知识已导致蛋白质数据库中沉积的16种结构，以及方法开发方面的尖端进展。经验表明，高分辨率膜蛋白结构的缺乏是结构测定管道中几种可识别瓶颈的结果，包括：（i）杂物原质的亚基共表达 蛋白质； （ii）真核膜蛋白的表达； （iii）确定稳定性和功能； （iv）识别（a）脂质在结构和结晶速率和（b）蛋白水解降解中的作用，这可以排除活性蛋白的纯化。将开发一系列新的表达方法（i）和（ii），并采用（ii）中的互补分析方法。问题（I-IV）导致形成良好的晶体的不确定性，这可能需要数周或数月的时间才能产生。早期检测晶体生长将大大减少晶体筛查所需的时间，并允许进入更大的结晶条件空间。我们描述了一种创新的方法，即SonicC（二阶非线性光学成像），用于对小至100 nm的初期蛋白质晶体的敏感和选择性检测，并在筛选平台中制备，其体积低至0.5 picoliter。它可以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。这种方法将极大地生成衍射质量的3维和二维晶体，​​这些晶体均在Surfo和Meso中产生。已经选择了一系列整体膜蛋白靶标，重点是ABC和异源蛋白。 （i）ABC：麦芽糖和核糖转运蛋白，ABCBL，ABCG2和 Sur-Kir6.2; （ii）非ABC：双精氨酸易位酶； KDP-ATPase; NADH脱氢酶。公共卫生相关性：整体膜蛋白控制细胞室之间的所有流量，膜本身的组装，对细胞的营养供应，其能量以及细胞与细胞外环境的通信。膜蛋白的原子结构对于理解人类疾病的分子基础以及药物的发展以对抗它们或改善其后果至关重要。