Fluorescence Anisotropy-based Macromolecule Crystallization Screening

基于荧光各向异性的高分子结晶筛选

• 批准号: 7479549
• 负责人: Marc Lee Pusey
• 金额: $ 10万
• 依托单位: IXPRESSGENES, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7479549
• 关键词: Anisotropy; Behavior; Biological Assay; Businesses; Condition; Crystallization; Data; Data Analyses; Data Collection; Databases; Diagnostic; Disease; Drops; Drug Delivery Systems; Drug Design; End Point; Fee-for-Service Plans; Fluorescence Anisotropy; Fluorescent Probes; Goals; Integral Membrane Protein; Label; Lead; Light; Measurement; Measures; Membrane Proteins; Methodology; Methods; Microscopy; Modeling; Numbers; Optics; Outcome; Performance; Phase; Precipitation; Probability; Process; Property; Proteins; Public Health; Range; Rate; Reporting; Roentgen Rays; Sampling; Screening Result; Screening procedure; Solutions; Source; Standards of Weights and Measures; Structure; Structure-Activity Relationship; Testing; Week; Work; base; experience; human disease; improved; instrument; macromolecule; novel strategies; research and development; research study; response; software development; structural genomics; success

DESCRIPTION (provided by applicant): Current practice is to set up trial crystallization screens and periodically review the results to see if a crystal or promising crystal-like precipitate has appeared a process that often takes weeks or months. Most outcomes are precipitated protein or clear drops, and the conditions that led to those results are dropped from further consideration. We propose an alternative screening approach, the self-association behavior of the target macromolecule as measured by fluorescence anisotropy as a diagnostic for the likelihood of crystallization under the test conditions. Dilute solution properties are known to be a diagnostic for crystallization (George and Wilson, 1994; George et al., 1997; Wilson et al., 1993; Wilson et al, 1996; Tessier et al., 2002; Tessier et al., 2003; Garcia et al., 2003a; Garcia et al., 2003b; Bloustine et al., 2003). Concentration vs. anisotropy data for a macromolecule-precipitant combination is proposed for determining the likelihood of that solution producing crystals. Preliminary data indicate that this approach can "find" lead crystallization conditions from solutions that give clear drops or precipitate in screening assays. The applications of this instrument and methodology will be to rapidly conduct crystallization screens within 2-3 hrs, using a minimum amount of protein (= 0.7 mg at 10 mg/mL), with a higher probability of finding lead conditions. Higher success rates will greatly facilitate structure-based drug design, particularly for target proteins that are difficult to obtain, and contribute to the understanding and treatment human disease. The Phase I proposal's objectives are to develop an instrument to make concentration vs. fluorescence anisotropy measurements, using = 100 ¿L of macromolecule solution for a 96 condition screen, and then validate the performance with extensive testing. Long range this instrument will be the basis for a macromolecule crystallization business operated on a fee for service basis. Experience with a breadboard "Phase 0" instrument has indicated where improvements can be made in the data collection and optics, and the initial Phase I work will be to assemble an improved instrument for making the anisotropy measurements. Subsequent testing will first be with model proteins, obtained commercially or from a local collaborating structural genomics effort, using manually prepared solutions. For each model protein the concentration vs. anisotropy data obtained will be compared with crystallization screens set up in parallel, to define the signature curves indicating crystallization or potential crystallization outcomes and the extended data range over which crystallization conditions can be recovered. All anisotropy-derived leads will be tested with optimization screens. Subsequent testing will be to challenge the methodology using previously uncrystallized soluble and membrane proteins from the same source. Projected subsequent Phase II efforts will be to reduce the amount of protein solution needed to = 10 nL, to robotically prepare the assay solutions, and to automate data analysis with software developed on the basis of the data obtained. PUBLIC HEALTH RELEVANCE: Successful crystallization and X-ray data analysis provides important three-dimensional information on the macromolecules structure-function relationship. Many proteins that are potential drug targets or key components in diseases are only available in trace quantities, or are difficult to obtain. This proposal is to develop a new approach to macromolecule crystallization, using a minimum amount of protein, and giving data that can subsequently be analyzed to determine those conditions which will give crystals and those that can be brought to crystallization conditions, thus giving a higher success rate.

描述（由申请人提供）：目前的做法是建立试验结晶筛选并定期审查结果，看看是否出现晶体或有希望的晶体状沉淀物，这一过程通常需要数周或数月的时间。大多数结果是沉淀的蛋白质或明确的下降，导致这些结果的条件从进一步考虑中删除。我们提出了一种替代的筛选方法，通过荧光各向异性测量的目标大分子的自缔合行为，作为测试条件下结晶的可能性的诊断。已知稀溶液性质是结晶的诊断（乔治和威尔逊，1994;乔治等人，1997; Wilson等人，1993; Wilson等人，1996; Tessier等人，2002; Tessier等人，2003; Garcia等人，2003 a; Garcia等人，2003 b; Bloustine等人，2003年）。浓度与各向异性数据的大分子沉淀剂组合提出确定的可能性，该解决方案产生晶体。初步数据表明，这种方法可以“找到”铅结晶条件下，从解决方案，给出明确的下降或沉淀在筛选试验。该仪器和方法的应用将是在2-3小时内快速进行结晶筛选，使用最少量的蛋白质（10 mg/mL时= 0.7 mg），发现铅条件的概率更高。更高的成功率将极大地促进基于结构的药物设计，特别是对于难以获得的靶蛋白，并有助于理解和治疗人类疾病。 第一阶段提案的目标是开发一种仪器，使用= 100 μ L的大分子溶液进行96条件筛选，进行浓度与荧光各向异性测量，然后通过广泛的测试验证性能。该仪器将成为高分子结晶业务的基础，该业务以服务收费为基础。试验板“阶段0”仪器的经验表明，在数据收集和光学方面可以作出改进，第一阶段的初步工作将是组装一个改进的仪器，进行各向异性测量。随后的测试将首先使用模型蛋白质，这些蛋白质是商业获得的或从当地合作的结构基因组学工作中获得的，使用手动制备的溶液。对于每种模型蛋白质，将获得的浓度与各向异性数据与平行设置的结晶筛选进行比较，以定义指示结晶或潜在结晶结果的特征曲线以及可以恢复结晶条件的扩展数据范围。所有各向异性衍生电极导线将通过优化筛选进行测试。后续检测将使用来自相同来源的先前未结晶的可溶性和膜蛋白挑战方法。预计随后的第II阶段工作将是将所需的蛋白质溶液量减少至≤ 10 nL，以机器人方式制备测定溶液，并使用基于所获得的数据开发的软件自动进行数据分析。 公共卫生关系：成功的结晶和X射线数据分析提供了重要的三维信息的大分子结构与功能的关系。许多蛋白质是潜在的药物靶点或疾病的关键成分，它们只能以微量获得，或难以获得。该提议是开发一种新的大分子结晶方法，使用最少量的蛋白质，并提供随后可以分析的数据，以确定将产生晶体的条件和可以达到结晶条件的条件，从而获得更高的成功率。