Biozentrum, University of Basel, Basel, Switzerland.
Methods Mol Biol. 2020;2127:397-419. doi: 10.1007/978-1-0716-0373-4_25.
The relationship of membrane protein function and the surrounding lipid bilayer goes far beyond simple hydrophobic interactions. At least from the 1980s, it is speculated that a certain fluid lipid state may be important not only for the lateral diffusion of membrane proteins (MPs) but also for modulating the catalytic activity of MPs (Lenaz. Bioscience Rep 7 (11):823-837, 1987). Indeed, acyl chain length, hydrophobic mismatch, and lipid headgroups are determinants for enzymatic and transport activities of MPs (Dumas et al. Biochemistry 39(16):4846-4854, 2000; Johannsson et al. Biochim Biophys Acta 641(2):416-421, 1981; Montecucco et al. FEBS Lett 144(1):145-148, 1982; Martens et al. Nat Struct Mol Biol 23(8):744-751, 2016). Moreover, it is speculated that changes in membrane lipid dynamics are important in the field of thermosensation (Vriens J, Nilius B, Voets T, Nat Rev Neurosci 15:573-589, 2014). Atomic insights into lipid-mediated modulation of membrane protein dynamics would therefore provide new insights with the potential to fundamentally extend our understanding on dynamic lipid-protein interdependencies.This chapter describes the expression and purification of nanodiscs assembled from membrane scaffold protein (MSP) as well as the expression and purification of the outer membrane protein X (OmpX). Subsequently, the incorporation of OmpX into MSP-derived nanodiscs is explained in detail. The chapter concludes with the setup of nuclear magnetic resonance (NMR) relaxation experiments and the extraction of relaxation rates for OmpX and the surrounding lipids.
膜蛋白功能与周围脂质双层的关系远不止于简单的疏水相互作用。至少从 20 世纪 80 年代开始，人们推测某种特定的流体脂质状态不仅对膜蛋白（MPs）的侧向扩散很重要，而且对调节 MPs 的催化活性也很重要（Lenaz. Bioscience Rep 7(11):823-837, 1987）。事实上，酰基链长、疏水性失配和脂质头部基团是决定 MPs 的酶和转运活性的因素（Dumas 等人，Biochemistry 39(16):4846-4854, 2000；Johannsson 等人，Biochim Biophys Acta 641(2):416-421, 1981；Montecucco 等人，FEBS Lett 144(1):145-148, 1982；Martens 等人，Nat Struct Mol Biol 23(8):744-751, 2016）。此外，人们推测膜脂动力学的变化在热感觉领域很重要（Vriens J, Nilius B, Voets T, Nat Rev Neurosci 15:573-589, 2014）。因此，原子水平上对脂质调节膜蛋白动力学的深入了解将为我们提供新的见解，并有潜力从根本上扩展我们对动态脂质-蛋白相互依赖关系的理解。
本章描述了膜支架蛋白（MSP）组装的纳米盘的表达和纯化，以及外膜蛋白 X（OmpX）的表达和纯化。随后，详细说明了 OmpX 掺入 MSP 衍生的纳米盘中的过程。最后介绍了核磁共振（NMR）弛豫实验的设置以及 OmpX 和周围脂质的弛豫率的提取。
