Innovative Methods for Membrane Protein Crystallization

膜蛋白结晶的创新方法

• 批准号: 7140615
• 负责人: Paul J. A. Kenis
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140615
• 关键词: NIH Roadmap Initiative tag; X ray crystallography; crystallization; high throughput technology; light scattering; membrane proteins; microfluidics; phase change; protein purification; technology /technique development

DESCRIPTION (provided by applicant): This proposal focuses on the creation and validation of novel crystallization platforms and methodology to substantially speed up the process of determining optimal crystallization conditions for membrane proteins. The crystallization platform will enable precise control of the path towards supersaturation, to identify crystal-producing conditions for different protein samples. In preliminary studies, we have demonstrated the principle with soluble proteins. We have developed a microscale vapor-diffusion-based device that, by precise control of the rate of evaporation and by continuous monitoring, leads to the rapid identification of crystal producing conditions. Once such conditions have been identified, the procedure is repeated for further optimization to improve crystal quality and growth. The crystallization platform offers advances over current devices used for screening crystallization conditions. (1) Control over the rate of solvent evaporation allows one to adjust the kinetics of attaining supersaturation and, therefore, crystallization events. (2) Setup of initial crystallization conditions in multiple wells will be formulated automatically using integrated large-scale microfluidic networks. (3) Through the use of microfluidics, precipitins and other reagents such as detergents can be added to each crystallization well during the course of the experiment at any time, adding a new dimension for exploration of crystallization events. To this end microfluidic technology will be used, which will also reduce the amount of protein needed. We hypothesize that use of the advanced controls integrated within the proposed crystallization platforms will increase the success rate of identifying crystallization condition for membrane proteins. Specific Aim 1: Demonstrate the utility of the current crystallization platform for membrane protein crystallization. We will validate our existing vapor-diffusion-evaporation platform for the identification of crystal-producing conditions for known and novel membrane proteins. Specific Aim 2: Design, fabricate and validate new and innovative microscale crystallization platforms for membrane protein crystallization. Aims include the complete control over evaporation rate, the automatic loading, the automatic creation of composition gradients across different compartments, as well as subsequent addition of precipitins and other reagents, as required for a high throughput screening platform for membrane protein crystallization. Specific Aim 3: Experimental study and model development to increase understanding of phase behavior of solutions used for membrane protein crystallization. Light and X-ray scattering studies will be performed to unravel the phase behavior; in particular the kinetics involved in phase changes, of lipid - protein solutions as typically used in membrane protein crystallization. We also intend to extend our recently developed population-balance based model capable of predicting kinetic parameters nucleation and growth of soluble proteins.

描述（由申请人提供）：该提案侧重于创建和验证新型结晶平台和方法，以大幅加快确定膜蛋白最佳结晶条件的过程。结晶平台将能够精确控制过饱和的路径，以确定不同蛋白质样品的晶体产生条件。在初步研究中，我们已经证明了可溶性蛋白质的原理。我们开发了一种基于微尺度蒸气扩散的装置，通过精确控制蒸发速率和连续监测，可以快速识别晶体生产条件。一旦确定了这样的条件，重复该过程以进一步优化以改善晶体质量和生长。 该结晶平台提供了优于当前用于筛选结晶条件的设备的优势。(1)对溶剂蒸发速率的控制允许调节获得过饱和的动力学，因此，结晶事件。(2)将使用集成的大规模微流体网络自动制定多个威尔斯孔中初始结晶条件的设置。(3)通过使用微流体，沉淀素和其他试剂如洗涤剂可以在实验过程中随时添加到每个结晶孔中，为探索结晶事件增加了新的维度。为此，将使用微流体技术，这也将减少所需的蛋白质量。我们假设，在拟定的结晶平台中集成先进控制的使用将提高识别膜蛋白结晶条件的成功率。 具体目标1：证明当前结晶平台对膜蛋白结晶的实用性。我们将验证我们现有的气相扩散蒸发平台，用于鉴定已知和新型膜蛋白的晶体产生条件。 具体目标2：设计、制造和验证用于膜蛋白结晶的新型和创新的微尺度结晶平台。目的包括完全控制蒸发速率、自动加载、跨不同隔室自动创建组成梯度以及随后添加沉淀素和其他试剂，这是膜蛋白结晶高通量筛选平台所需的。 具体目标3：实验研究和模型开发，以增加对用于膜蛋白结晶的溶液相行为的理解。将进行光和X射线散射研究以揭示相行为;特别是涉及膜蛋白结晶中通常使用的脂质-蛋白质溶液的相变的动力学。我们还打算扩展我们最近开发的人口平衡为基础的模型，能够预测动力学参数成核和可溶性蛋白质的生长。