Solution NMR Spectroscopy for the Determination of Structures of Membrane Proteins in a Lipid Environment

Abstract

NMR spectroscopy has harnessed the recent technical advances to emerge as a competitive, elegant, and eminently viable technique for determining the solution structures of membrane proteins at the level of atomic resolution. Once a good level of cell-based or cell-free expression and purification of a suitably sized membrane protein has been achieved, then NMR offers a combination of several versatile strategies, for example choice of appropriate deuterated or nondeuterated detergents, temperature, and ionic strength; isotope labeling with 2H, 13C, 15N, with or without protonation of Ile (δ1), Leu, and Val methyl protons; combinatorial labeling or unlabeling of specific amino acids; TROSY based-, nonuniform sampling (NUS) based-, and other NMR experiments; measurement of residual dipolar couplings using stretched polyacrylamide gels or DNA nanotubes; spin labeling and paramagnetic relaxation enhancements (PRE). Strategic combinations of these advancements together with availability of highly sensitive cryogenically cooled-probes equipped high-field NMR spectrometers (up to 1GHz 1H frequency) have allowed the perseverant investigator to successfully overcome several of the conventional pitfalls associated with the NMR technique and membrane proteins, viz., low sensitivity, poor sample stability, spectral crowding, and a limited number of NOEs and other constraints for structure calculations. This has resulted in an unprecedented growth in the number of successfully determined NMR structures of large and complex membrane proteins over the last two decades, and this technique now holds great promise for the structure determination of an ever larger body of membrane proteins.