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）：本提案旨在解决可作为药物靶点和医学用途的完整膜蛋白的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成熟专业知识，这导致了16种结构沉积在蛋白质数据库中，以及方法开发的前沿进展。经验表明，高分辨率膜蛋白结构的缺乏是结构测定管道中几个可识别的瓶颈的结果，包括：(i)异亚基原核生物亚基的共表达