Fluorescence Anisotropy-based Macromolecule Crystallization Screening

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，看看是否出现了晶体或有希望的晶体样沉淀物，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清澈的液滴，导致这些结果的条件被排除在进一步的考虑之外。我们正在开发一种替代筛选方法，通过荧光各向异性测量目标大分子的自结合行为，作为在测试条件下结晶可能性的诊断。稀溶液性质被认为是结晶的诊断指标（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等，2003）。一种大分子-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，强度与浓度数据也是一个强有力的指标。第一阶段获得的数据表明，这种方法可以在筛选分析中从给出清晰滴或沉淀的溶液中“找到”铅的结晶条件。待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），具有较高的发现铅条件的可能性。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。II期提案的目标是大幅改进目前的仪器和方法，从目前所需的4.5 mg蛋白质/96条件筛选提高到0.03 mg，并通过广泛的测试验证该方法。这将成为按服务收费的高分子结晶业务的基础。第一阶段仪器的经验表明，在板设置过程、数据收集光学和电子方面可以进行改进，而第二阶段的初始工作将是实施这些改进。随后的测试将首先使用模型蛋白。对于每种模型蛋白，所获得的浓度、各向异性和强度数据将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。所有各向异性衍生的引线都将使用优化屏幕进行测试。随后的测试将使用来自同一来源的先前未结晶的可溶性蛋白和膜蛋白来挑战方法。该模型和测试蛋白数据将用于后续数据分析软件的开发。预计第三阶段的工作包括开发不完全因子筛选，可以充分利用该方法获得的定量数据。

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.