Fluorescence Anisotropy-based Macromolecule Crystallization Screening

描述(申请人提供)：目前的做法是建立试验结晶屏幕，并定期审查结果，以确定是否出现了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。大多数结果是沉淀的蛋白质或明显的液滴，导致这些结果的条件被排除在进一步考虑之外。我们正在开发另一种筛选方法，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(George和Wilson，1994；George等，1997；Wilson等，1993；Wilson等，1996；Tessier等，2002；Tessier等，2003；Garcia等，2003a；Garcia等，2003b；Bloustine等，2003)。大分子-沉淀剂组合的浓度与各向异性数据最初是为了确定该溶液产生晶体的可能性而提出的，尽管基于第一阶段的结果，浓度与浓度的数据也是一个强有力的指标。在第一阶段获得的数据表明，这种方法可以从筛选分析中提供清晰液滴或沉淀的溶液中找到铅结晶条件。将要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毫克)，发现铅条件的可能性更高。更高的成功率将极大地促进基于结构的药物设计，特别是难以获得的靶蛋白的药物设计，并有助于理解和治疗人类疾病。第二阶段提案的目标是大幅改进目前的仪器和方法，从目前需要的4.5毫克蛋白质/96条件筛查进展到0.03毫克蛋白质/96条件筛查，并通过广泛的测试验证这一方法。这将是以收费服务为基础运营的高分子结晶业务的基础。第一阶段仪器的经验表明，可以在板设置过程、数据采集光学和电子学方面进行改进，而第二阶段的初始工作将是实施这些改进。随后的测试将首先用模型蛋白质进行。对于每个模型蛋白质，获得的浓度与各向异性和强度数据将与平行设置的结晶屏幕进行比较，以定义指示结晶或潜在结晶结果的签名曲线，以及可以恢复结晶条件的扩展数据范围。所有各向异性引线都将通过优化筛选进行测试。随后的测试将挑战这一方法，使用来自同一来源的先前未结晶的可溶性和膜蛋白。该模型和测试蛋白质数据将用于后续数据分析软件的开发。预计的第三阶段工作包括开发能够充分利用该方法获得的定量数据的不完全析因筛选。 与公众健康相关：成功的结晶和X射线数据分析提供了关于大分子结构-功能关系的重要三维信息。许多作为潜在药物靶点或疾病关键成分的蛋白质只能微量获得，或者很难获得。这一建议是继续开发一种新的大分子结晶方法，使用最少的蛋白质，并提供定量数据，随后可以分析这些数据，以确定哪些条件将产生晶体，哪些条件可以使其结晶，从而提供更高的成功率。

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.