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。我们提出了另一种筛选方法，通过荧光各向异性测量目标大分子的自缔合行为，以此来诊断在测试条件下结晶的可能性。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)。提出了一种大分子-沉淀剂组合的浓度与各向异性数据，以确定该溶液产生晶体的可能性。初步数据表明，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。这种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。 第一阶段提案的目标是开发一种仪器来进行浓度与荧光各向异性的测量，使用=100°L的高分子溶液进行96条件筛选，然后通过广泛的测试验证性能。长远来说，这台仪器将成为按服务收费经营的高分子结晶业务的基础。使用面包板“0相”仪器的经验表明，可以在数据收集和光学方面进行改进，第一阶段的初始工作将是组装一种改进的仪器，用于进行各向异性测量。随后的测试将首先使用人工准备的解决方案，对商业上或从当地合作的结构基因组学工作中获得的模型蛋白质进行测试。对于每个模型蛋白质，获得的浓度与各向异性的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。预计随后的第二阶段工作将是减少所需的蛋白质溶液的数量，以=10nL，自动制备化验溶液，并利用根据所获得的数据开发的软件实现数据分析的自动化。 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。这一建议是开发一种新的高分子结晶方法，使用最少的蛋白质，并提供数据，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 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.