Nanodiscs to study membrane proteins

膜蛋白占所有生物细胞含量的近30%。尽管它们对生命系统非常重要，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，膜蛋白的体外操作需要研究人员使用脂泡或洗涤剂胶束等方法来模拟原始生物膜的性质。然而，这些环境通常会降低蛋白质的活性，或者在更糟糕的情况下，由于膜的异质性和蛋白质的聚集而产生难以分析的结果。为了帮助绕过这些困难，一种名为纳米盘或纳米脂肪颗粒的技术正在开发中，该技术将蛋白质整合到小的(几个纳米大的)盘状膜片中。这项技术是新颖的，使研究人员能够应用各种新的实验技术，因为这些颗粒稳定、均匀且可溶于水。这项技术使我的实验室能够了解蛋白质跨细菌膜转运的基本过程。在我们提出的研究计划中，我们将在三个不同的应用中进一步开发纳米盘，这将有助于(I)了解专门的膜转运体如何允许糖等营养物质穿过细菌膜，(Ii)识别蛋白质为了提取和运输胆固醇而需要与脂类建立分子联系，以及(Iii)使用适应于纳米盘的最新蛋白质组学工具发现细菌细胞膜中发生的蛋白质相互作用网络。这些目标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。我们使用纳米级脂双分子层的研究具有很强的下游生物技术应用潜力。它还将培训我的学生和博士后研究人员掌握广泛的技能，从而为他们在学术或工业环境中的专业职业生涯做好准备。

Membrane proteins constitute nearly 30% of the cell content for all living organisms. Despite their great importance to life systems, 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 researchers to use methods such as lipid vesicles or detergent micelles that mimic the properties of the original biological membrane. However, these environments often reduce the activity of proteins, or in worse cases, produce results that are problematic to analyze due to membrane heterogeneity and protein aggregation. To help to circumvent the difficulties, a technology known as Nanodiscs, or Nanolipoparticles, is being developed to incorporate proteins into small (a few nanometer large) discoidal patches of membrane. This technology is novel and enable researchers to apply a variety of new experimental techniques because these particles are stable, homogeneous and water-soluble. The technology allowed my laboratory to understand the fundamental process of protein translocation across the bacterial membrane. In our proposed research program, we will further exploit the Nanodiscs in three different applications that will serve (i) to understand how specialized membrane transporters allow nutrients such as sugars to cross the bacterial membrane, (ii) to identify the molecular contacts that proteins need to establish with lipids in order to extract and transport cholesterol, and (iii) to discover the network of protein interactions taking place in the bacterial cell envelope using the latest tools of proteomics adapted to Nanodiscs. Each of these goals will contribute important new information to key cellular process that occurs at the membrane interface. Our research using the nanoscale lipid bilayers has strong potential for downstream biotechnological applications. It will also train my students and post-doctoral researchers with a wide range of skills, thereby preparing them for professional careers in either academic or industrial settings.