Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics

Membrane proteins, and especially G-protein coupled receptors (GPCRs), are increasingly important targets of structural biology studies due to their involvement in many biomedically critical pathways in humans. These proteins are often highly dynamic and thus benefit from studies by NMR spectroscopy in parallel with complementary crystallographic and cryo-EM analyses. However, such studies are often complicated by a range of practical concerns, including challenges in preparing suitably isotopically labeled membrane protein samples, large sizes of protein/detergent or protein/lipid complexes, and limitations on sample concentrations and stabilities. Here we describe our approach to addressing these challenges via the use of simple eukaryotic expression systems and modified NMR experiments, using the human adenosine A_2A receptor as an example. Protocols are provided for the preparation of U-²H (¹³C,¹H-Ile δ1)-labeled membrane proteins from overexpression in the methylotrophic yeast Pichia pastoris, as well as techniques for studying the fast ns–ps sidechain dynamics of the methyl groups of such samples. We believe that, with the proper optimization, these protocols should be generalizable to other GPCRs and human membrane proteins.