Fluorescent Methods for Protein Crystallization

蛋白质结晶的荧光方法

• 批准号: 6951243
• 负责人: Marc Lee Pusey
• 金额: $ 12万
• 依托单位: U.S. MARSHALL SPACE FLIGHT CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6951243
• 关键词: X ray crystallography; biomedical automation; crystallization; fluorescent dye /probe; functional /structural genomics; high throughput technology; macromolecule; molecular probes; protein purification; protein structure; protein structure function; structural biology; synchrotrons; technology /technique development

DESCRIPTION (provided by applicant): This proposal addresses the methodology employed for obtaining protein crystals for structural genomics or other purposes. Understanding the relationship between macromolecule structure and function is critical to an understanding of the molecular aspects of physiological functions essential to human health. This proposal is to test and develop the use of trace labeling of macromolecules undergoing crystallization screening for analyzing the results obtained. This proposal is to: 1) Demonstrate that the trace fluorescently labeled protein can be used to facilitate the crystal screening process, and determine a practical modification range for use in visual crystal detection and scoring of experimental results; 2) Develop facile protocols for making fluorescent derivatives that will readily fit within high-throughput purification and preparation procedures; 3) Determine the usable range for labeling macromolecules, from the minimal amount for detection to the maximum level, where X-ray data or crystallization is affected; 4) Test the utility of the labeled crystals for automated crystal alignment at synchrotron beamlines. The primary means of attaining these goals will be by comparative crystallization trials followed by X-ray analysis of the crystals obtained. Trace fluorescent labeled proteins will be provided to a synchrotron beamline (Argonne Nat. Lab.) for use in developing automated alignment methods. By covalently modifying a subpopulation of a protein solution with a fluorescent probe, the labeled population will add to a growing crystal as a microheterogeneous growth unit. The probe will concentrate in the crystal relative to the solution, and under fluorescent conditions they will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity may also be used to distinguish previously obscure crystallization lead conditions. The fluorescent labeled crystals will also emit with sufficient intensity to aid in the automation of crystal mounting using relatively low cost optics, further increasing throughput at synchrotrons. While this proposal focuses on a visual approach, it is anticipated that success will subsequently lead to development of automated methods of analysis, more suitable for high throughput application.

描述（由申请人提供）：该提案提出了用于获得用于结构基因组学或其他目的的蛋白质晶体的方法。了解大分子结构和功能之间的关系对于理解对人类健康至关重要的生理功能的分子方面至关重要。该提案旨在测试和开发使用微量标记进行结晶筛选的大分子，以分析所获得的结果。该提案的目的是：1）证明痕量荧光标记蛋白可用于促进晶体筛选过程，并确定用于可视晶体检测和实验结果评分的实际修饰范围； 2) 开发用于制备荧光衍生物的简便方案，使其易于适合高通量纯化和制备程序； 3）确定标记大分子的可用范围，从检测的最小量到影响X射线数据或结晶的最大水平； 4) 测试标记晶体在同步加速器光束线处自动晶体对准的实用性。实现这些目标的主要方法是比较结晶试验，然后对所得晶体进行 X 射线分析。痕量荧光标记蛋白质将提供给同步加速器光束线（阿贡国家实验室），用于开发自动对准方法。通过用荧光探针共价修饰蛋白质溶液的亚群，标记的群体将作为微异质生长单元添加到正在生长的晶体中。探针将相对于溶液集中在晶体中，并且在荧光条件下，它们将在黑暗背景下显示为明亮的物体。由于晶体堆积比无定形沉淀物更致密，因此荧光强度也可用于区分以前模糊的结晶先导条件。荧光标记的晶体还将发出足够的强度，以帮助使用相对低成本的光学器件实现晶体安装的自动化，从而进一步提高同步加速器的吞吐量。虽然该提案侧重于视觉方法，但预计成功将随后导致自动化分析方法的开发，更适合高通量应用。