Assignment of oriented sample NMR resonances from a three transmembrane helix protein

Oriented sample solid state NMR techniques have been routinely employed to determine the structures of membrane proteins with one or two transmembrane helices. For larger proteins the technique has been limited by spectral resolution and lack of assignment strategies. Here, a strategy for resonance assignment is devised and applied to a three transmembrane helix protein. Sequence specific assignments for all labeled transmembrane amino acid sites are obtained, which provide a set of orientational restraints and helix orientations in the bilayer. Our experiments expand the utility of solid state NMR in membrane protein structure characterization to three transmembrane helix proteins and represent a straightforward strategy for routinely characterizing multiple transmembrane helix protein structures.