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: 7998996
• 负责人: Marc Lee Pusey
• 金额: $ 37万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7998996
• 关键词: Anisotropy; Appearance; Behavior; Biological Assay; Businesses; Collection; Computer software; Crystallization; Data; Data Analyses; Data Collection; Development; Diagnostic; Diffusion; Disease; Drops; Drug Delivery Systems; Drug Design; Electronics; Fee-for-Service Plans; Fluorescence; Fluorescence Anisotropy; Fluorescent Probes; Genes; Goals; Label; Lead; Marketing; Measures; Membrane Proteins; Methodology; Methods; Metric; Microfluidics; Modeling; Modification; Optics; Outcome; Performance; Phase; Probability; Process; Property; Protein Structure Initiative; Proteins; Reliance; Research; Resolution; Roentgen Rays; Screening Result; Screening procedure; Services; Small Business Innovation Research Grant; Solutions; Source; Structure; Structure-Activity Relationship; System; Testing; Time; Work; base; commercial application; data acquisition; design; experience; gene synthesis; hepatocyte growth factor-regulated tyrosine kinase substrate; human disease; improved; instrument; instrumentation; macromolecule; novel strategies; protein function; protocol development; public health relevance; research and development; response; software development; success; technological innovation; two-photon

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 removed from further consideration. We are developing 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 was originally proposed for determining the likelihood of that solution producing crystals, although based on Phase I results intensity vs. concentration data is also a strong indicator. Data acquired in Phase I shows that this approach can "find" lead crystallization conditions from solutions that give clear drops or precipitate in screening assays. The applications of the instrument and methodology to be developed will be to rapidly conduct crystallization screens within 2-24 hrs, using a minimum amount of protein ( 0.03 mg), 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 II proposal's objectives are to substantially improve the present instrument and methodology, progressing from the currently required 4.5 mg to 0.03 mg of protein/96 condition screen, and validate this method with extensive testing. This will be the basis for a macromolecule crystallization business operated on a fee-for-service basis. Experience with the Phase I instrument has indicated where improvements can be made in the plate set-up process, data collection optics, and electronics and the initial Phase II work will be to implement those improvements. Subsequent testing will first be with model proteins. For each model protein the concentration vs. anisotropy and intensity 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. The model and test protein data will be used in the subsequent development of software for data analysis. Projected Phase III efforts include developing incomplete factorial screens that can make full use of the quantitative data obtained by this method. 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 continue development of a new approach to macromolecule crystallization, using a minimum amount of protein, and giving quantitative 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的考虑中删除。我们正在开发一种替代的筛选方法，通过荧光各向异性测量目标大分子的自缔合行为，作为测试条件下结晶可能性的诊断。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年; Garcia等人，2003 a; Garcia等人，2003 b; Bloustine等人，2003年）。最初提出大分子-沉淀剂组合的浓度与各向异性数据用于确定溶液产生晶体的可能性，尽管基于阶段I结果，强度与浓度数据也是一个强指标。在第一阶段获得的数据表明，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。将开发的仪器和方法的应用将是在2-24小时内快速进行结晶筛选，使用最少量的蛋白质（0.03 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。 第二阶段提案的目标是实质性地改进目前的仪器和方法，从目前所需的4.5 mg蛋白质/96条件筛选进展到0.03 mg蛋白质/96条件筛选，并通过广泛的测试验证该方法。这将是一个高分子结晶业务的基础上经营的收费服务的基础。第一阶段仪器的经验表明，在板设置过程中，数据收集光学器件和电子器件可以进行改进，第二阶段的初步工作将是实施这些改进。随后的测试将首先使用模型蛋白质。对于每种模型蛋白质，将获得的浓度与各向异性和强度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。模型和测试蛋白数据将用于数据分析软件的后续开发。预计第三阶段的努力包括开发不完全的因子筛选，可以充分利用这种方法获得的定量数据。 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。该提议是继续开发一种新的高分子结晶方法，使用最少量的蛋白质，并提供定量数据，随后可以分析这些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。