Development of a Computerized Multi-Station Protein Crystallization Apparatus

9317981费格尔森这项建议是为了开发一种多工位计算机控制的设备，用于生长高质量的生物大分子(蛋白质)单晶，适用于通过X射线衍射进行结构测定。生长合适的晶体1的困难一直是限制蛋白质结构数量的主要因素之一，这些结构已经完全确定2。蛋白质分子结构的测定已经成为现代生物学和药理学的一个重要部分，其潜力几乎没有被挖掘出来。所提出的装置的开发是为了生长具有高度内部完备性的晶体，这将有助于结构确定的过程。为了生长出高质量的晶体，有必要在结晶过程中分别控制成核相和生长相。我们实验室以前的工作已经证明，通过控制溶液中较小区域的温度，可以控制蛋白质溶液中晶体的成核。随后的生长通过改变生长细胞周围的环境温度来控制。这项技术，热成核法。已被用于成核和生长无机和蛋白质材料的晶体3，4。热成核依赖于成核区和溶液主体的温度的独立控制。它的使用仅限于那些在大约0到35℃的温度范围内对溶解度具有一定温度依赖性的蛋白质，因此适用于大量蛋白质。该计划的目的是开发一种用户友好的、计算机控制的多工位设备，以利用热成核原理生长蛋白质晶体。生长模块将在之前开发的研究仪器的基础上缩小尺寸，并将使用热电加热和制冷。新的设计将强调晶体收获和蛋白质溶液更换的便利性。实际生长细胞的大小将与蛋白质结晶中使用的小溶液体积一致。温度感应将内置到每个生长模块中。该模块还将包括成核检测系统，该系统将允许从计算机控制的成核程序切换到适当的生长程序。成核检测将基于静态光散射技术5。该装置的开发将从生长模块的设计、建造和测试开始。与此同时，将开发成核探测器。这两个单元将被集成到一个模块中。至此，与计算机的接口和计算机程序的开发将开始。当使用多个结晶系统在计算机控制下对单个组件进行彻底测试后，将构建和测试多个单元装置。仪器开发的每个阶段的结果将被用于优化最终的仪器。拟议计划的目标将是将这项技术开发成一种用户友好的半自动设备，使生化工作者能够在最少的操作员交互的情况下，重复生长用于X射线结构测定的高质量蛋白质单晶。

9317981 Feigelson This proposal is for the development of a multi-station, computer controlled apparatus for the growth of high quality, single crystals of biological macromolecules (proteins) suitable for use in structure determinations by x- ray diffraction. The difficulties in growing suitable crystals 1 has been one of the major factors limiting the number of protein structures which have been completely determined 2 . The determination of the molecular structure of proteins has become an important segment of modern biology and pharmacology and its potential has been barely tapped 2 . The development of the proposed apparatus is directed toward the growth of crystals with a high degree of internal perfection which will facilitate the process of structure determination. In order to growth the highest quality crystals, it is necessary to separately control the nucleation and growth phases during crystallization. Previous work in our laboratory has demonstrated that the nucleation of crystals in protein solutions can be controlled by controlling the temperature in a small region of the solution. The subsequent growth was controlled by changing the ambient temperature around the growth cell. This technique, Thermonucleation. has been used to nucleate and grow crystals of both inorganic and protein materials 3,4 . Thermonucleation relies on the independent control of the temperatures in both the nucleating region and the bulk of the solution. Its use is limited to those proteins which have some temperature dependence of solubility within the temperature range of approximately 0 to 35 C and thus is applicable to a large number of proteins. The intent of the proposed program is to develop a user friendly, computer controlled, multi-station apparatus to grow protein crystals using the principle of Thermonucleation. The growth module will be downsized from the research instrument previously developed and will use thermoclectric heating and cooling . The new design will emphasize ease of crystal harvesting and changing of protein solutions. The actual growth cell will be of a size consistent with the small solution volumes used in protein crystallization. Temperature sensing will be built into each growth module. The module will also incorporate a nucleation detection system which will allow switching from a computer controlled nucleation program to the appropriate growth program. Nucleation detection will be based on static light scattering techniques 5 . The development of the apparatus will begin with the design, construction and testing of the growth module. Concurrent with this, the nucleation detector will be developed. These two units will be integrated into a single module. At this point, interfacing with the computer and computer program development will begin. When the single module has been thoroughly tested under computer control using a number of crystallizing systems multiple unit apparatus will be constructed and tested. Results at each stage of the apparatus development will be used to optimize the final apparatus. The goal of the proposed program will be to develop this technology into a user friendly, semi-automated apparatus which will allow biochemical worker to reproducibly grow high quality single crystals of proteins for x-ray structure determinations with a minimum of operator interaction.