Purifying Membrane Proteins within Native Lipid Bilayers

膜蛋白(MPS)是最重要但也是最具挑战性的生物分子。 解决方案。对MPS的研究的一个主要问题是对从其本土环境中提取的要求， 膜。几十年来，化学和MP群落贡献了不同的膜 有助于增溶和稳定MPS的模拟物。尽管它们有广泛的应用，但这些膜仿真剂 不要完全复制含有数百种不同脂质的MPS的天然双层设置。这是一个 根本问题，因为脂双层和特定的脂相互作用对结构组装和 多个MPS的功能。尽管如此，关键的挑战仍然是如何孤立和稳定脆弱的议员和 MP络合物对其构象、低聚物组装和功能几乎没有干扰，任务是 然而，对结构、功能和药物相互作用的研究至关重要。要克服这些重要的 在MP研究中的挑战，我们建议开发一种策略，直接在小块中丰富或纯化MPS 细胞膜。我们的建议利用了两种流行的膜试剂的良好性能， 即小分子洗涤剂和两亲性聚合物，同时避免它们在1) 在大多数MP过敏中，洗涤剂是有效的，不可避免地用于分散细胞膜，但洗涤剂- 溶解的膜贴片是高度动态和不稳定的结构；和2)两亲性聚合物 支架可以包裹和稳定中央双层贴片，但大多数用于稳定MPS的聚合物是无效的 分散细胞膜。为了解决这一困境，我们提出了一种全新的聚合物设计，将小分子 膜结构域通过原位交联剂，分散在细胞膜上。要建立 这种方法的可行性，我们将探索化学用于洗涤剂交联剂的增溶和稳定小 细胞膜片(目标1)，并验证化学工具在纯化不同的MPS(目标2)。如果 如果成功，我们的开发将提供一种全新的、多功能的方法来准备MPS 原生环境。实现这一目标以及新工具的开发将对 MP研究的许多领域，其中需要分离MPS。

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.