Innovative Methods for Membrane Protein Crystallization

描述(由申请人提供)：本提案侧重于创建和验证新的结晶平台和方法，以大大加快确定膜蛋白最佳结晶条件的过程。结晶平台将实现对过饱和路径的精确控制，以确定不同蛋白质样品的晶体产生条件。在初步研究中，我们已经用可溶性蛋白质证明了这一原理。我们已经开发了一种基于微尺度蒸气扩散的设备，通过精确控制蒸发速度和连续监测，可以快速识别晶体的生产条件。一旦确定了这样的条件，就重复这个过程，进行进一步的优化，以提高晶体质量和生长。 结晶平台提供了比现有用于筛选结晶条件的设备更先进的设备。(1)对溶剂挥发速度的控制使人们能够调整达到过饱和的动力学，从而调节结晶事件。(2)利用集成的大规模微流控网络，自动设定多个井的初始结晶条件。(3)通过使用微流体，可以在实验过程中随时向每个结晶井中添加沉淀物和洗涤剂等试剂，为结晶事件的探索增加了一个新的维度。为此，将使用微流控技术，这也将减少所需的蛋白质数量。我们假设，使用集成在所建议的结晶平台中的高级控制将提高识别膜蛋白结晶条件的成功率。 具体目标1：展示现有的膜蛋白结晶平台的实用性。我们将验证我们现有的蒸气扩散-蒸发平台，以确定已知和新的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的和创新的微尺度结晶平台。目的包括完全控制蒸发速度、自动加载、跨不同隔室自动创建组成梯度，以及随后添加沉淀物和其他试剂，这是高通量膜蛋白结晶筛选平台所需的。 具体目标3：实验研究和模型开发，以增加对用于膜蛋白质结晶的溶液的相行为的了解。将进行光和X射线散射研究，以揭示膜蛋白结晶中通常使用的脂蛋白溶液的相行为，特别是涉及相变的动力学。我们还打算扩展我们最近开发的基于种群平衡的模型，该模型能够预测可溶蛋白质的成核和生长的动力学参数。

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.