Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins

Journal of the American Chemical Society (Impact Factor: 11.44). 01/2013; 135(5). DOI: 10.1021/ja310901f
Source: PubMed

ABSTRACT Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent containing environments. While nanodiscs can offer a near native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin we show that our smaller nanodiscs can also be beneficial for the structural characteri-zation of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for any given membrane protein while still providing a functional environment. In addi-tion to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of inter-actions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biological relevant interactions.

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    • "Nevertheless, in the absence of a functional test in vitro, high-quality spectroscopic signals do not prove that the protein is in its native conformation (Poget and Girvin 2007; Zhou and Cross 2013; Catoire et al. 2014). For instance, OmpX exhibits various backbone 15 N/ 1 H N chemical shifts depending on the surfactant used (Fernández et al. 2001; Lee et al. 2008; Hagn et al. 2013). These variations are unlikely to be due to changes in the transmembrane electronic environment, given that, whatever the surfactant used, the amino acids pointing toward the membrane face mostly CH n moieties. "
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