NMR studies of membrane proteins typically use micelles or bicelles as membrane surrogates. These have the disadvantage of a destabilizing effect of the detergents, in particular when the membrane proteins have water-soluble domains or interact with soluble proteins, such as GPCRs interacting with heterotrimeric G proteins and ligands. Therefore, we embraced the use of phospholipid nanodiscs, which are patches of phospholipid bilayer surrounded by two copies of a membrane-scaffolding protein (Msp1), which is derived from apolipoprotein A1. We had shown that the Voltage-Dependent Anion Channel (VDAC1) can be inserted into nanodiscs and analysed both in EM images and in NMR spectra. However, embedding was heterogeneous and prevented detailed NMR analysis. To address this problem and adapt nanodiscs to different size membrane proteins, and we could determine an NMR structure of the OmpX model protein in an 8.1 nm nanodisc. Unfortunately, the diameter distribution of these nanodiscs is rather wide. Thus, we developed procedures to covalently circularize the scaffolding protein and make nanodiscs of exactly defined diameters. We could extend the cND sizes from as low as 8 nm to as high as 50 nm each exhibiting a very narrow size distribution. We can insert membrane protein into the covalently circularized nanodiscs, record NMR spectra for smaller systems and obtain negative stain or cryo EM images from nanodisc-bound membrane proteins. We pursued applications from small mitochondrial membrane proteins to GPCRs, GPCR/G-protein complexes and larger systems. To avoid amide back-exchange problems on membrane proteins that cannot be refolded we do not deuterate and use <sup>15</sup>N detected TROSY spectroscopy.<br/>

Engineering nanodiscs for membrane protein studies

254th National Meeting (2017)

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