Development of a Computerized Multi-Station Protein Crystallization Apparatus

9317981 Feigelson该提案旨在开发一种多站、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。 生长合适的晶体的困难1一直是限制蛋白质结构数量的主要因素之一，蛋白质结构已经完全确定2。 蛋白质分子结构的测定已成为现代生物学和药理学的重要组成部分，其潜力尚未得到充分挖掘。 所提出的设备的发展是针对具有高度的内部完善性的晶体的生长，这将有助于结构测定的过程。 为了生长最高质量的晶体，有必要在结晶过程中分别控制成核和生长阶段。 我们实验室以前的工作已经证明，蛋白质溶液中晶体的成核可以通过控制溶液中一个小区域的温度来控制。 通过改变生长池周围的环境温度来控制随后的生长。 这种技术，热核。已经被用于无机和蛋白质材料3，4的成核和生长晶体。 热成核依赖于成核区域和溶液本体中的温度的独立控制。 它的应用限于在约0 - 35 ℃的温度范围内溶解度具有一定温度依赖性的那些蛋白质，因此可应用于大量蛋白质。 该项目的目的是开发一种用户友好的，计算机控制的，多站的设备，使用热成核的原则来生长蛋白质晶体。 生长模块将从以前开发的研究仪器缩小，并将使用热电加热和冷却。新的设计将强调易于晶体收获和蛋白质溶液的改变。实际生长池的大小将与蛋白质结晶中使用的小溶液体积一致。温度传感将内置于每个生长模块中。该模块还将包含一个成核检测系统，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、构造和测试开始开始。 与此同时，将开发成核检测器。 这两个单元将合并为一个单元。 此时，将开始与计算机和计算机程序开发接口。 当单个模块在计算机控制下使用多个结晶系统进行彻底测试时，将构建和测试多个单元装置。 在设备开发的每个阶段的结果将用于优化最终的设备。 拟议计划的目标是将这项技术开发成一种用户友好的半自动化设备，使生物化学工作者能够以最少的操作员交互可重复地生长高质量的蛋白质单晶，用于X射线结构测定。

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.