Testing Feasibility of SlipChip Workflow for Membrane Protein Crystallization

描述（由申请人提供）：膜蛋白对多种生理过程至关重要，其功能是化学受体、运输通道和信号转导。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、稳定性差等瓶颈，获得膜蛋白的衍射晶体仍然是困难的；更大参数空间的搜索；难以获得足以确定结构的衍射。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，可以潜在地实现这些目标。它已经应用于复杂的方案，如酶分析，免疫分析和PCR分析。PI已经证明，在玻璃中制造的SlipChip可以用于处理和结晶膜蛋白，并获得可溶性蛋白的晶体和高分辨率结构。然而，许多适用于可溶性蛋白的技术不适用于膜蛋白；此外，结晶工作流程中单个步骤的许多优雅解决方案（例如建立自由界面扩散）会在其他地方产生额外的瓶颈（例如在放大或晶体提取中产生问题）。本一期提案的目标是建立该创新在膜蛋白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)简单的加载，可以在不需要任何设备的情况下可靠地测量一系列溶液的nL体积，到目标2)性能，从填充到孵育和观察，通过廉价的成型技术用塑料制成的芯片，到目标3)强大的提取和晶体衍射方法。实现这些具体目标将坚定地确立SlipChip膜蛋白结晶技术的可行性和可行性，并将降低该领域二期工作的技术风险，二期工作将整合许多步骤，如浓缩、分离、洗涤剂交换、纯化、并将中相形成为一个芯片，该芯片接受纯化柱上的一小部分，并进行复杂的操作，以产生衍射质量的膜蛋白晶体。

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.