Multi-Level Optimization of Membrane Proteins for Crystallography

用于晶体学的膜蛋白的多级优化

• 批准号: 8536322
• 负责人: MARK E. DUMONT
• 金额: $ 118.29万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8536322
• 关键词: Affect; Automation; Behavior; Benign; Bicarbonates; Bile Acids; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Calorimetry; Carbon Dioxide; Cell Maintenance; Cell Proliferation; Cell physiology; Cells; Chemicals; Cloning; Cocrystallography; Code; Codon Nucleotides; Collaborations; Communities; Community Outreach; Complex; Crystallization; Crystallography; Detergents; Development; Disease; Drug Design; Endocannabinoids; Engineering; Enzymes; Erythrocytes; Escherichia coli; Family; Flow Cytometry; Fluorescent Probes; G-Protein-Coupled Receptors; Gel Chromatography; Genes; Genome; Growth; Heterogeneity; Homeostasis; Human; Human Resources; Insecta; Institutes; Integral Membrane Protein; Ion Transport; Ions; Knowledge; Label; Laboratories; Libraries; Ligands; Lipids; Mammals; Measurement; Mediating; Medical; Membrane; Membrane Proteins; Methods; Mind; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Molecular Structure; Mutate; Nutrient; Oils; Oligopeptides; Organism; Orthologous Gene; Oxygen; Pharmaceutical Preparations; Phase; Physiology; Play; Post-Translational Protein Processing; Principal Investigator; Procedures; Process; Production; Property; Protein Family; Protein Structure Initiative; Proteins; Proteolysis; Protocols documentation; Publishing; Reagent; Recording of previous events; Refractive Indices; Research Infrastructure; Research Personnel; Role; Signal Transduction; Solubility; Solutions; Solvents; Sphingolipids; Staging; Structure; System; Techniques; Technology; Testing; Thermodynamics; Time; Titrations; Transmembrane Transport; Ursidae Family; Validation; Variant; Work; Yeasts; base; design; expression cloning; high throughput screening; improved; intercellular communication; interest; large scale production; light scattering; member; novel strategies; pH Homeostasis; prenyl; programs; protein S precursor; protein complex; protein function; protein structure; receptor function; screening; structural biology; structural genomics; success; technology development; three dimensional structure; uptake; yeast genetics

Transmembrane proteins (TMPs) comprise more than 25% of the protein-coding potential of most genomes. They also play central roles in cell and organismal physiology and are the targets of a large fraction of all clinically useful drugs. However, there is a huge deficit in our knowledge of the structure and function of TMPs in comparison to soluble proteins. This can be primarily attributed to the substantial roadblocks generally encountered in applying x-ray crystallography to TMPs. This application brings together three independent PIs with diverse backgrounds in the molecular biology, biochemistry, biophysics, and structural biology, specifically of membrane proteins, to create a pipeline for TMP structure determination. A central tenet of the pipeline is the need to discriminate at the earliest possible stage in production between protein targets that are amenable to structure determination and those that are not. With this in mind, we propose to target families of orthologous, paralogous, and mutated proteins, carrying multiple variants through the early stages of purification and characterization so as to maximize the chances of advancing the most tractable members of a target family to the point of successful crystallization and diffraction. The project makes use of existing cloning and expression protocols for the bacterial and yeast expression systems that are most amenable to parallel expression strategies, but will use bacculovirus expression for some proteins. Expression testing will be conducted using small-scale growths; multiple forms of a given target will be produced at an intermediate scale to allow characterization using an existing high-throughput screen for detergent compatibility, biophysical and biochemical characterization and small-scale exploratory crystallization trials. Only the most promising candidates from intermediate scale analysis will be carried forward to large scale production for high-throughput crystallization screening using the facilities of the Hauptman-Woodward Institute. Additional screening using lipidic cubic phases will be conducted as needed. In addition to proteins expected from the PSI Network, initial structure determination efforts will target three classes of proteins: certain classes of transmembrane transporters, enzymes involved in lipid synthesis and lipid attachment to proteins, and complexes of seven-transmembrane segment proteins, including GPCRs, with single pass chaperone-like accessory proteins. The project also seeks to develop improved technologies for increasing levels of expression of functional TMPs in yeast, new approaches for specific fluorescent labeling of unpurified proteins, and the development of improved methods for biophysical characterization and screening of protein detergent complexes.

跨膜蛋白(TMP)占大多数基因组蛋白质编码能力的25%以上。它们在细胞和生物体生理学中也发挥着核心作用，是所有临床有用药物的一大部分靶标。然而，与可溶性蛋白质相比，我们对TMPS的结构和功能的了解存在着巨大的不足。这主要可以归因于在将X射线结晶学应用于TMP时通常遇到的实质性障碍。这项应用将分子生物学、生物化学、生物物理学和结构生物学(特别是膜蛋白)领域的三个具有不同背景的独立PI结合在一起，创建了一条测定TMP结构的管道。该管道的一个中心原则是，需要在生产的最早阶段区分适合于结构确定的蛋白质靶标和不适合结构确定的蛋白质靶标。考虑到这一点，我们建议针对直源、平行和突变蛋白家族，在纯化和鉴定的早期阶段携带多个变体，以便最大限度地将目标家族中最易处理的成员推进到成功结晶和衍射的点。该项目利用现有的细菌和酵母表达系统的克隆和表达协议，这些系统最适合并行表达策略，但将使用杆状病毒表达一些蛋白质。将使用小规模培养进行表达测试；将在中等规模生产多种形式的特定靶标，以便使用现有的高通量筛选进行洗涤剂配伍性、生物物理和生化表征以及小规模探索性结晶试验。只有来自中等规模分析的最有希望的候选者才会利用Hauptman-Woodward研究所的设施进行大规模生产，以进行高通量结晶筛选。根据需要，将使用脂类立方相进行额外的筛查。除了预期来自PSI网络的蛋白质外，初步结构确定工作将针对三类蛋白质：某些类型的跨膜转运蛋白、参与脂质合成和与蛋白质结合的酶，以及包括GPCRs在内的七跨膜段蛋白质与单程伴侣样辅助蛋白的复合体。该项目还寻求开发改进的技术，以提高功能性TMPS在酵母中的表达水平，开发用于未纯化蛋白质的特定荧光标记的新方法，以及开发改进的生物物理表征和蛋白质洗涤剂复合体的筛选方法。