Laurence Matthew J, Carpenter Timothy S, Laurence Ted A, Coleman Matthew A, Shelby Megan, Liu Chao
Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
Materials Science Division, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
Membranes (Basel). 2022 Mar 31;12(4):392. doi: 10.3390/membranes12040392.
Proteins embedded in biological membranes perform essential functions in all organisms, serving as receptors, transporters, channels, cell adhesion molecules, and other supporting cellular roles. These membrane proteins comprise ~30% of all human proteins and are the targets of ~60% of FDA-approved drugs, yet their extensive characterization using established biochemical and biophysical methods has continued to be elusive due to challenges associated with the purification of these insoluble proteins. In response, the development of nanodisc techniques, such as nanolipoprotein particles (NLPs) and styrene maleic acid polymers (SMALPs), allowed membrane proteins to be expressed and isolated in solution as part of lipid bilayer rafts with defined, consistent nanometer sizes and compositions, thus enabling solution-based measurements. Fluorescence correlation spectroscopy (FCS) is a relatively simple yet powerful optical microscopy-based technique that yields quantitative biophysical information, such as diffusion kinetics and concentrations, about individual or interacting species in solution. Here, we first summarize current nanodisc techniques and FCS fundamentals. We then provide a focused review of studies that employed FCS in combination with nanodisc technology to investigate a handful of membrane proteins, including bacteriorhodopsin, bacterial division protein ZipA, bacterial membrane insertases SecYEG and YidC, type III secretion protein YopB, yeast cell wall stress sensor Wsc1, epidermal growth factor receptor (EGFR), ABC transporters, and several G protein-coupled receptors (GPCRs).
嵌入生物膜的蛋白质在所有生物体中发挥着重要功能，充当受体、转运蛋白、通道、细胞粘附分子以及其他支持细胞功能的角色。这些膜蛋白约占人类所有蛋白质的30%，并且是约60%的FDA批准药物的靶点，然而，由于与这些不溶性蛋白质纯化相关的挑战，使用既定的生化和生物物理方法对其进行广泛表征仍然难以实现。作为回应，纳米盘技术的发展，如纳米脂蛋白颗粒（NLPs）和苯乙烯马来酸聚合物（SMALPs），使得膜蛋白能够在溶液中作为具有明确、一致纳米尺寸和组成的脂质双层筏的一部分进行表达和分离，从而实现基于溶液的测量。荧光相关光谱（FCS）是一种相对简单但强大的基于光学显微镜的技术，它能产生关于溶液中单个或相互作用物种的定量生物物理信息，如扩散动力学和浓度。在这里，我们首先总结当前的纳米盘技术和FCS基本原理。然后，我们重点综述了一些研究，这些研究将FCS与纳米盘技术相结合，以研究少数几种膜蛋白，包括细菌视紫红质、细菌分裂蛋白ZipA、细菌膜插入酶SecYEG和YidC、III型分泌蛋白YopB、酵母细胞壁应激传感器Wsc1、表皮生长因子受体（EGFR）、ABC转运蛋白以及几种G蛋白偶联受体（GPCRs）。
