Nanodiscs to study membrane proteins

膜蛋白占所有生物体细胞含量的近30%。尽管它们对生命系统非常重要，但科学家们仍在努力研究这些大分子在功能上的活性形式，并与实验相兼容。传统上，在体外操作膜蛋白需要研究人员使用脂质囊泡或洗涤剂胶束等方法来模拟原始生物膜的特性。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，由于膜的非均质性和蛋白质聚集，产生难以分析的结果。为了帮助克服这些困难，一种被称为纳米圆盘或纳米脂粒的技术正在被开发出来，它可以将蛋白质整合到小的（几纳米大小的）膜盘状斑块中。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，因为这些颗粒是稳定的，均匀的和水溶性的。这项技术使我的实验室了解了蛋白质在细菌膜上易位的基本过程。在我们提出的研究计划中，我们将进一步在三种不同的应用中开发纳米圆盘，这将有助于(i)了解特殊的膜转运体如何允许糖等营养物质穿过细菌膜，（ii）确定蛋白质与脂质建立分子接触以提取和运输胆固醇，（iii）利用最新的蛋白质组学工具，发现细菌细胞包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提供重要的新信息。我们使用纳米级脂质双分子层的研究在下游生物技术应用方面具有很强的潜力。它还将培养我的学生和博士后研究人员广泛的技能，从而为他们在学术或工业环境中的职业生涯做好准备。

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.