NMR structural studies of membrane proteins

Abstract

Membrane proteins are responsible for many significant biological functions including those of enzymes, channels, pumps, receptors, and anchors. Solid-state NMR spectroscopy can be used to study oriented and unoriented samples of membrane proteins in bilayers because their reorientation rates are slower than 10\sp{-4} sec. A solid-state NMR probe with a flat coil was constructed and used for experiments to determine the structures of the membrane bound forms of peptides and proteins oriented between a pair of glass plates. Proteins and peptides were obtained by expression in bacteria and by solid-phase peptide synthesis and were specifically, selectively, and uniformly \sp{15}N labeled. Ligand gated ion channel peptide M2 subunit, bacteriophage Pf1(Class II) coat protein, fd (Class I) coat protein, and M13 procoat protein which has a 23 leader sequence were studied. The pore forming segment M2 of the ligand gated ion channel super family is a trans-membrane peptide oriented perpendicular to the bilayer. Pf1 coat protein in membrane bilayers has two helices. The amphipathic helix is parallel to the plane of the bilayer and the hydrophobic helix is perpendicular to the plane of the bilayer. There is a mobile loop between the two helices and the protein has mobile NH\sb2- and -COOH termini. fd coat protein in membrane bilayers also has two helices and mobile NH\sb2- and -COOH termini. The turn between the two helices is structured, even though there is evidence of Tyr 21 mobility on slow time scales. The leader sequence of M13 procoat protein has a mobile NH\sb2-terminus, a hydrophobic region which is parallel to the bilayer, and a mobile cleavage site for leader peptidase. The dynamics of the mature region of the M13 procoat protein are almost the same as for fd coat protein