NMR Spectroscopy as a Tool to Provide Mechanistic Clues About Protein Function and Disease Pathogenesis

Nuclear magnetic resonance (NMR) has become an important technique for determining the three-dimensional (3D) structure of biological macromolecules. Since 1985, it has been used to determine the structures of approximately 8,000 proteins, 1,000 DNA/RNA complexes and 180 protein/nucleic acid complexes (http://www.pdb.org/pdb/ statistics/holdings.do). At first, NMR was limited to relatively small and soluble proteins or protein domains. The study of large proteins was hindered by the presence of overlapping large peaks in the NMR spectra. This has been in part alleviated by the introduction of isotope (2H, 15N, 13C) labeling and multidimensional (3D, 4D) experiments (Sattler et al., 1999). By using these techniques, it is now possible to study proteins up to 40 kDa. For instance, the 3D solution structure of the maltodextrin-binding protein (41kDa) has been recently solved using NMR (Madl et al., 2009; Figure 1A). Comprehensive NMR studies of integral membrane proteins in solution have long been impaired by substantial problems of sample preparation, including the inability to produce sufficient quantities of isotopically labelled protein as well as the difficulties associated with the limited thermal stability, sample heterogeneity and short lifetimes of such proteins. However, NMR allows investigation of the very conformational mobility that to a large extent interferes with the process of crystallization of membrane proteins. Thus, by focusing on proteins with a sufficient expression yield and screening sample and detergent conditions in a microtiter-plate format, it has been possible to determine 71 structures of integral membrane proteins corresponding to 49 unique proteins (http://www.drorlist.com/ nmr/MPNMR.html). The first NMR structure determination of a detergent-solubilized seven-helix transmembrane (7TM) protein, the phototaxis receptor sensory rhodopsin II (pSRII) from Natronomonas pharaonis, was recently reported to illustrate that NMR can provide structures of large membrane proteins (Gautier et al., 2010; Figure 1B). The challenge is now to apply similar techniques to the study of other 7TM proteins, including G-protein coupled receptors (GPCRs) that represent the most important class of targets for current therapeutic agents. These proteins can only be