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以与实验兼容的功能活性形式研究这些大分子。传统上，体外膜蛋白的操作需要研究人员使用诸如脂质囊泡或去污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，或者在更坏的情况下，由于膜异质性和蛋白质聚集而产生分析有问题的结果。 为了帮助克服这些困难，正在开发一种称为Nanodisks或Nanolipoparticles的技术，将蛋白质整合到小的（几纳米大）圆盘状膜中。 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能够了解蛋白质穿过细菌膜的基本过程。 在我们提出的研究计划中，我们将进一步利用纳米盘在三个不同的应用，这将有助于（i）了解专门的膜转运蛋白如何允许营养物质，如糖穿过细菌膜，（ii）确定蛋白质需要建立与脂质的分子接触，以提取和运输胆固醇，以及（iii）使用适用于Nanodisks的蛋白质组学的最新工具来发现细菌细胞包膜中发生的蛋白质相互作用的网络。 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。 我们的研究使用的纳米脂质双层具有强大的潜力，下游生物技术的应用。 它还将培养我的学生和博士后研究人员具有广泛的技能，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.