Testing Feasibility of SlipChip Workflow for Membrane Protein Crystallization

描述(申请人提供)：膜蛋白在多种生理过程中起关键作用，起化学受体、运输通道和信号转导的作用。据估计，超过一半的药物靶点是膜蛋白。膜蛋白的结构知识具有很高的生物医学意义。膜蛋白结晶是获取膜蛋白结构信息的重要途径。然而，由于膜蛋白的数量少、稳定性差、寻找更大的参数空间以及难以获得足够的衍射物来确定结构，获得膜蛋白的衍射晶仍然是困难的。目前还没有技术可以解决所有的需求和瓶颈。SlipChip是一项有吸引力的创新技术，有可能实现这些目标。它已经被应用于复杂的程序，如酶分析、免疫分析和聚合酶链式反应分析。等电点表明，玻璃中的SlipChip可用于处理和结晶膜蛋白，并获得可溶蛋白的晶体和高分辨率结构。然而，许多适用于可溶性蛋白质的技术在膜蛋白质方面失败了；此外，结晶工作流程中单一步骤的许多优雅解决方案(例如，建立自由界面扩散)在其他地方造成了额外的瓶颈(例如，在放大或晶体提取方面造成问题)。这项第一阶段建议的目标是确定这项创新在膜蛋白质领域的可行性和适用性，同时测试结晶工作流程中上游和下游步骤的可行性：从目标1)简单加载，无需任何设备即可可靠地测量出一系列解决方案的NL体积，到目标2)通过廉价的模塑技术制造塑料芯片的性能，从灌装到孵化和观察，到目标3)稳健的晶体提取和衍射方法。实现这些特定目标将坚定地确立SlipChip技术用于膜蛋白结晶的可行性和可行性，并将降低这一领域第二阶段工作的技术风险，该阶段将把浓缩、分离、洗涤剂交换、纯化和形成中间相的许多步骤整合到一个芯片中，该芯片接受纯化柱下的一部分，并进行复杂的操作以生产膜蛋白的衍射级晶体。 与公众健康相关：膜蛋白的结构知识对公众健康具有非常高的生物医学意义。膜蛋白的结晶是获取其结构信息的重要途径，但由于存在许多瓶颈，结晶过程十分困难。这项提案描述了一种滑动芯片技术，以解决膜蛋白结晶的未满足需求和瓶颈。

DESCRIPTION (provided by applicant): Membrane proteins are crucial to a wide variety of physiological processes, functioning as chemical receptors, transport channels and signal transducers. It is estimated that over half of drug targets are membrane proteins. Structural knowledge of membrane proteins is of very high biomedical significance. Crystallization of membrane proteins is an important approach to obtaining structural information of membrane proteins. However, obtaining diffracting crystals of membrane proteins is still difficult due to a number of bottlenecks, including low quantity and poor stability of membrane proteins; search of larger parameter space; and difficulty to obtaining diffraction sufficient to determine structures. No technology is currently available to address all of the needs and bottlenecks. SlipChip is an attractive and innovative technology that can potentially achieve these goals. It has been already applied to complex protocols such as enzyme assays, immunoassays, and PCR assays. The PI has demonstrated that SlipChip fabricated in glass can be used to handle and crystallize membrane proteins, and obtain crystals and high-resolution structures of soluble proteins. Yet, many technologies applicable to soluble proteins fail with membrane proteins; in addition, many elegant solutions to a single step in the crystallization workflow (e.g. establishing free interface diffusion) create additional bottlenecks elsewhere (e.g. creating problems in scale-up or crystal extraction). The goal of this Phase I proposal is to establish the feasibility and applicability of this innovation in the area of membrane proteins, while testing the feasibility of upstream and downstream steps in the crystallization workflow: from Aim 1) simple loading that will meter out nL volumes robustly for a range of solutions without the need for any equipment, to Aim 2) performance, from filling to incubation and observation, of chips made in plastic by inexpensive molding techniques, to Aim 3) robust methods of extraction and diffraction of crystals. Reaching these specific aims would firmly establish feasibility and viability of SlipChip technology for membrane protein crystallization, and would reduce the technical risk of Phase II work in this area, which would integrate many of the steps of concentration, separation, detergent exchange, purification, and formation of meso-phases into a single chip that accepts a fraction off a purification column and performs complex manipulations to produce diffraction-quality crystals of membrane proteins. PUBLIC HEALTH RELEVANCE: Structural knowledge of membrane proteins is of very high biomedical significance to public health. Crystallization of membrane proteins is an important approach to obtaining their structural information, but is difficult due to a number of bottlenecks. This proposal describes a SlipChip technology to address unmet needs and bottlenecks for membrane protein crystallization.