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

Uniformly N-15-labeled samples of membrane proteins with helices aligned pa rallel 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 improve d in three-dimensional H-1 chemical shift/H-1-N-15 dipolar coupling/N-15 ch emical shift correlation spectra. The 23-residue antibiotic peptide magaini n and a 54-residue polypeptide corresponding to the cytoplasmic domain of t he HIV-1 accessory protein Vpu are used as examples. Both polypeptides cons ist almost entirely of alpha-helices, with their axes aligned parallel to t he membrane surface. The measurement of three orientationally dependent fre quencies for Val17 of magainin enabled the three-dimensional orientation of this helical peptide to be determined in the lipid bilayer. (C) 2000 Acade mic Press.