Tools for High-Throughput Protein Crystallization and Structure Determination

描述（由申请人提供）：我们对生命分子机制的理解越来越多地基于我们对蛋白质、核酸、病毒和生物分子复合物的三维结构的了解。结构提供了对功能（或故障）的洞察，并为现代药物发现和分子医学提供了起点。生物分子结构通常是用结晶生物分子的x射线晶体学来确定的。在过去的十年中，高通量方法已经被引入，这些方法已经自动化了蛋白质表达，纯化，结晶和晶体学的许多方面，并且降低了确定每个结构的成本。然而，蛋白质晶体的生长和收获仍然是从基因到三维分子结构以及从结构到药物治疗的主要瓶颈。第二阶段STTR提案的重点是开发和商业化传统和高通量结晶以及晶体收集和x射线数据收集的改进方法。康奈尔大学（Cornell University）对液体接触线与表面相互作用的研究，带来了一种简单的技术，可以精确地定义液滴的位置，并将液滴牢牢地固定在这些位置上，而不管它们的化学成分如何。这项技术为蛋白质结晶板的新方法奠定了基础，该方法消除了传统板的限液孔。这些新板有望提供精确控制滴位置和形状，从而产生更可重复的结晶动力学和简化的图像分析。它们将允许在单个平板上悬挂和静滴生长可溶性和膜蛋白，并在低背景下进行原位光学、紫外线和x射线分析。它们将满足对优化板的关键需求，以便于筛选和结晶结果的x射线检查，并允许原位结构测定。它们将与所有现有的滴胶和印版处理硬件兼容，降低市场进入壁垒。该项目将继续对这些板进行科学和商业开发，并探索滴钉技术的其他生物医学应用。在第一阶段，我们成功开发并商业化了几种用于晶体检索和x射线数据收集的新工具。我们将在第二阶段继续这一发展，重点是改进微晶体学、自动样品安装、x射线束对准和能量测量的工具。总之，这些技术应该对高通量结构基因组学和药物发现工作的生产力产生重大影响。

DESCRIPTION (provided by applicant): Our understanding of the molecular mechanisms of life is increasingly based upon our knowledge of the three dimensional structure of proteins, nucleic acids, viruses and biomolecular complexes. Structure provides insight into function (or malfunction), and provides a starting point for modern drug discovery and molecular medicine. Biomolecular structures are most often determined using X-ray crystallography of crystallized biomolecules. Over the last decade, high-throughput methods have been introduced that have automated many aspects of protein expression, purification, crystallization and crystallography, and that have lowered the cost per structure determined. However, the growth and harvesting of protein crystals remains a major bottleneck in the pipeline from gene to three-dimensional molecular structure and from structure to pharmaceutical therapy. This Phase II STTR proposal is focused on developing and commercializing improved methods for conventional and high-throughput crystallization and for crystal harvesting and X-ray data collection. An examination at Cornell University of how liquid contact lines interact with surfaces has led to a simple technology for precisely defining the positions of dispensed liquid drops and firmly holding them to those positions, regardless of their chemical composition. This technology forms the basis for a new approach to protein crystallization plates that eliminates the liquid-confining wells of conventional plates. These new plates promise to provide precise control over drop position and shape, resulting in more reproducible crystallization kinetics and simplified image analysis. They will allow hanging and sitting drop growth of soluble and membrane proteins using a single plate, and in situ optical, UV and X-ray analysis with low background. They will meet a critical need for plates optimized for easy X-ray examination of screening and crystallization outcomes and also allow in situ structure determination. They will be compatible with all existing drop dispensing and plate handling hardware, lowering barriers to market entry. This project will continue the scientific and commercial development of these plates and explore other biomedical applications of drop pinning technology. In Phase I we successfully developed and commercialized several new tools for crystal retrieval and X-ray data collection. We will continue this development in Phase II, focusing on improved tools for microcrystallography, for automated sample mounting, and for X-ray beam alignment and energy measurement. Together, these technologies should have significant impact on the productivity of high- throughput structural genomics and drug discovery efforts. PUBLIC HEALTH RELEVANCE: Our understanding of the molecular mechanisms of life is increasingly based upon our knowledge of the three dimensional structure of proteins, nucleic acids and viruses. Structure provides insight into function (or malfunction), and provides a starting point for modern drug discovery and molecular medicine. This project will develop and commercialize new technologies for use in determining the protein structures by X-ray crystallography that promise to speed up the progression from gene to pharmaceutical therapy.