Three-dimensional solid-state NMR spectroscopy is essential for resolution of resonances from in-plane residues in uniformly 15N-labeled helical membrane proteins in oriented lipid bilayers

Uniformly N-15-labeled samples of membrane proteins with helices aligned parallel to the membrane surface give two-dimensional PISEMA spectra that are highly overlapped due to limited dispersions of H-1-N-15 dipolar coupling and N-15 chemical shift frequencies. However, resolution is greatly improved in three-dimensional H-1 chemical shift/H-1-N-15 dipolar coupling/N-15 chemical shift correlation spectra. The 23-residue antibiotic peptide magainin and a 54-residue polypeptide corresponding to the cytoplasmic domain of the HIV-1 accessory protein Vpu are used as examples. Both polypeptides consist almost entirely of alpha-helices, with their axes aligned parallel to the membrane surface. The measurement of three orientationally dependent frequencies for Val17 of magainin enabled the three-dimensional orientation of this helical peptide to be determined in the lipid bilayer. (C) 2000 Academic Press.