Innovative Methods for Membrane Protein Crystalliza(RMI)

膜蛋白结晶（RMI）的创新方法

• 批准号: 7011041
• 负责人: Paul J. A. Kenis
• 金额: $ 18.13万
• 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 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7011041
• 关键词: 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）：该提案重点介绍了新型结晶平台和方法的创建和验证，以实质上加快确定膜蛋白的最佳结晶条件的过程。结晶平台将能够精确控制过饱和路径，以确定不同蛋白质样品的结晶条件。在初步研究中，我们证明了可溶性蛋白的原理。我们已经开发了一种基于微观蒸气 - 扩散的设备，通过精确控制蒸发速率并通过连续监测，可以快速鉴定晶体产生条件。一旦确定了此类条件，重复该过程以进一步优化以提高晶体质量和生长。 结晶平台比用于筛选结晶条件的当前设备提供了进步。 （1）控制溶剂蒸发速率，可以调整获得过饱和的动力学，从而调整结晶事件。 （2）将使用集成的大规模微流体网络自动制定多个井中初始结晶条件的设置。 （3）通过使用微流体，沉淀蛋白和其他试剂（例如洗涤剂）在实验过程中可以很好地添加到每个结晶中，从而增加了探索结晶事件的新维度。为此，将使用微流体技术，这还将减少所需的蛋白质量。我们假设在提出的结晶平台中集成的高级控件的使用将增加识别膜蛋白结晶条件的成功率。 特定目标1：演示膜蛋白结晶的当前结晶平台的实用性。我们将验证现有的蒸汽扩散蒸发平台，以鉴定已知和新型膜蛋白的晶体产生条件。 特定目标2：设计，制造和验证用于膜蛋白结晶的新的和创新的微观晶体结晶平台。目的包括对蒸发率的完整控制，自动载荷，跨不同隔室的组成梯度的自动创建以及随后添加沉淀蛋白和其他试剂，这是膜蛋白结晶的高吞吐量筛选平台所需的所需。 特定目标3：实验研究和模型开发，以增加对用于膜蛋白结晶溶液的相行为的理解。将进行光和X射线散射研究以揭示相行为。尤其是在膜蛋白结晶中通常使用的脂质 - 蛋白质溶液中涉及的动力学。我们还打算扩展我们最近开发的基于人群平衡的模型，能够预测可溶性蛋白质的动力学参数成核和生长。