Abstract
Membrane proteins perform many important cellular functions. Historically, structural studies of these proteins have been conducted in detergent preparations and synthetic lipid bilayers. More recently, magic-angle-spinning (MAS) solid-state NMR has been employed to analyze membrane proteins in native membrane environments, but resonance assignments with this technique remain challenging due to limited spectral resolution and high resonance degeneracy. To tackle this issue, we combine reverse labeling of amino acids, frequency-selective dipolar dephasing, and NMR difference spectroscopy. These methods have resulted in nearly complete resonance assignments of the transmembrane domain of human LR11 (SorLA) protein in E. coli membranes. To reduce background signals from E. coli lipids and proteins and improve spectral sensitivity, we effectively utilize amylose affinity chromatography to prepare membrane vesicles when MBP is included as a fusion partner in the expression construct.