Membrane proteins: structure - function - methods of analysis

利用原生纳米盘的力量 膜蛋白存在于所有生物膜中。它们具有许多基本功能，其表面位置使其成为制药和生物技术行业的理想药物靶标。尽管它们非常重要，但科学家们仍在努力以一种与实验兼容的功能活性形式来研究这些大分子。传统上，在体外操纵膜蛋白需要使用不能模拟原始生物膜性质的洗涤剂。即使是最轻微的疾病，也会导致蛋白质不稳定或失去蛋白质活性，此外还会破坏蛋白质与蛋白质之间的联系。 苯乙烯-马来酸(SMA)脂质颗粒，也被称为天然或SMA纳米盘，是一种从膜中提取蛋白质的新工具，破坏程度最小。在SMA-纳米盘的生成过程中，苯乙烯部分插入到脂酰链之间，形成一个围绕着一小块脂双层(直径约10-15 nm)的“手环”。通过这种方式，膜蛋白和蛋白质复合体以及它们的天然脂类可以从天然膜中提取和纯化。除了保留天然脂质环境和蛋白质-蛋白质相互作用的固有优势外，由于这些颗粒稳定且可溶于水，因此可以开发出广泛的应用。 天然纳米盘是膜生物学领域一个令人振奋的新发展。在这项提案中，我们将以三种不同的方式利用本地纳米盘的力量。我们将1)使用基于蛋白质组的方法来鉴定和分离在洗涤剂中不稳定的新的膜蛋白复合体，2)构建SMA-纳米盘库以识别几乎任何给定配体的膜受体(S)，以及3)开发一种允许快速鉴定任何类型细胞的膜蛋白周围的特定脂类的方案。这些应用中的每一个都将为发生在膜界面的关键细胞过程贡献重要的新信息。此外，由于膜蛋白在天然状态下变得更容易获得，我们的工作可能会激发制药部门的兴趣，特别是以前被认为太难有效制造用于下游应用(如药物发现)的膜蛋白靶标。

Exploiting the power of the native nanodisc Membrane proteins are found in all biological membranes. They have many essential functions and their surface location make them ideal drug targets in pharmaceutical and biotechnology industries. Despite their great importance, scientists still struggle to study these macromolecules in a functionally active form that is compatible with experimentation. The manipulation of membrane proteins in vitro traditionally requires the use of detergents that fail to mimic the properties of the original biological membrane. Even the mildest ones lead to protein destabilization or loss of protein activity, in addition to destroying protein-protein associations. The styrene-maleic acid (SMA) lipid particle, also termed native- or SMA-nanodisc, is a new tool to extract proteins from membrane with minimal amount of disruption. During SMA-nanodisc generation, the styrene moieties intercalate between the lipid acyl chains to form a “bracelet” which encircles a small patch of lipid bilayer (~10-15 nm diameter). In this manner, membrane proteins and also protein complexes, together with their native lipids, can be extracted and purified from the native membrane. In addition to the inherent advantage of conserving the native lipid environment and protein-protein interactions, a wide variety of applications can be developed because these particles are stable and water-soluble. The native nanodisc is an exciting new development in the field of membrane biology. In this proposal, we will exploit the power of native nanodiscs in three different ways. We will 1) employ a proteomic-based method to identify and isolate novel membrane protein complexes that are unstable in detergent, 2) construct a library of SMA-nanodiscs to identify membrane receptor(s) of virtually any given ligand, and 3) develop a protocol to allow rapid identification of specific lipids surrounding membrane proteins of any type of cell. Each of these applications will contribute important new information to key cellular process that occurs at the membrane interface. Furthermore, since membrane proteins are becoming easier to obtain in their native state, our work might stimulate interest from the pharmaceutical sector, especially for membrane protein targets previously considered too difficult to manufacture efficiently for downstream applications such as drug discovery.