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从进一步的考虑中删除。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）。提出了大分子-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“发现”铅结晶条件的溶液给出明确滴或沉淀筛选分析。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（= 0.7 mg/mL, 10 mg/mL），具有更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。

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.