Purifying Membrane Proteins within Native Lipid Bilayers

纯化天然脂质双层内的膜蛋白

• 批准号: 10132363
• 负责人: Qinghai Zhang
• 金额: $ 22.19万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10132363
• 关键词: Address; Affect; Affinity; Antibodies; Area; Binding; Biological; Biological Process; Buffers; Cell membrane; Chemical Structure; Chemicals; Chemistry; Cholesterol; Cholic Acids; Chromatography; Collaborations; Communities; Complex; Detergents; Development; Dissociation; Drug Interactions; Drug Screening; Drug Targeting; Environment; Face; Feedback; G-Protein-Coupled Receptors; Genes; Goals; Human; In Situ; Ligands; Lipid Bilayers; Lipids; Liposomes; Membrane; Membrane Lipids; Membrane Proteins; Methodology; Micelles; Molecular Conformation; Particle Size; Performance; Phase; Polymers; Preparation; Process; Property; Proteins; Reagent; Reporting; Research; Resolution; Steroids; Structure; Styrenes; System; Technology; Testing; amphiphilicity; biophysical techniques; cost; crosslink; design; drug discovery; functional group; improved; maleic acid; membrane model; mimetics; nanodisk; nanosized; new technology; particle; preservation; prevent; protein complex; protein purification; protein structure; reconstitution; scaffold; small molecule; tool; tool development

Membrane proteins (MPs) are important but arguably the most challenging biological molecules to study in solution. One major concern with studies of MPs is the requirement for extraction from their native environment, the membrane. Over several decades the chemistry and MP communities have contributed diverse membrane mimetics that help to solubilize and stabilize MPs. Despite their broad applications, these membrane mimetics do not fully replicate the native bilayer setting of MPs which contains hundreds of diverse lipids. This is a fundamental issue because lipid bilayers and specific lipid interactions are crucial to the structural assembly and function of many MPs. Notwithstanding, key challenges remain as to how to isolate and stabilize fragile MPs and MP complexes with little disturbance to their conformation, oligomer assembly, and function, tasks that are nevertheless crucial to structural, functional and drug interaction studies. To overcome these important challenges in MP research, we propose to develop a strategy to directly enrich or purify MPs within small patches of cell membranes. Our proposal exploits the favorable properties of two types of popular membrane reagents, i.e. small molecule detergents and amphiphilic polymers, and meanwhile avoid their shortcomings in that 1) detergent is effective and inevitably used to disperse cell membranes in most MP pruritions, but the detergent- solubilized membrane patches are highly dynamic and unstable structures; and 2) an amphiphilic polymer scaffold may entrap and stabilize a central bilayer patch, but most polymers used to stabilize MPs are ineffective to disperse cell membranes. To resolve the dilemma, we propose an ex novo polymer design to encircle small membrane domains by crosslinking detergents in situ, upon the dispersion of cell membranes. To establish the feasibility of this approach, we will explore chemistry for detergent crosslinking to solubilize and stabilize small patches of cell membranes (Aim 1) and validate chemical tools in the purification of diverse MPs (Aim 2). If successful, our development will provide a completely new and versatile approach to preparing MPs in nearly native environment. Achieving this goal together with new tool development will have a far-reaching impact on many areas of MP research in which the isolation of MPs is needed.

膜蛋白（MP）是重要的，但可以说是最具挑战性的生物分子研究， 溶液对MP研究的一个主要关注点是需要从其原生环境中提取， 膜。几十年来，化学和MP社区已经贡献了各种膜 有助于溶解和稳定MP的模拟物。尽管这些膜模拟物具有广泛的应用， 不能完全复制含有数百种不同脂质的MP的天然双层设置。这是一 这是一个根本问题，因为脂质双层和特定的脂质相互作用对结构组装至关重要， 许多议员的作用。尽管如此，如何隔离和稳定脆弱的议员， MP复合物的构象，寡聚体组装和功能几乎没有干扰， 然而对于结构、功能和药物相互作用研究至关重要。为了克服这些重要的 在MP研究的挑战，我们建议开发一种策略，直接富集或纯化MP内小补丁 指细胞膜。我们的建议利用了两种常用膜试剂的有利特性， 即小分子洗涤剂和两亲性聚合物，同时避免了它们缺点：1） 洗涤剂是有效的，不可避免地用于分散细胞膜在大多数MP pruries，但洗涤剂- 溶解的膜贴片是高度动态和不稳定的结构;和2）两亲聚合物 支架可以捕获并稳定中心双层片，但大多数用于稳定MP的聚合物是无效的 来分散细胞膜。为了解决这个难题，我们提出了一种从头聚合物设计， 膜域通过交联洗涤剂原位，在细胞膜的分散。建立 这种方法的可行性，我们将探索化学洗涤剂交联溶解和稳定小 细胞膜补丁（目标1）和验证化学工具在不同的MP纯化（目标2）。如果 成功，我们的发展将提供一个全新的和多功能的方法来准备国会议员在近 原生环境实现这一目标与新工具的开发一起将对 许多需要隔离议员的议员研究领域。