The secondary structure and alignment of hydrophobic model peptides in phosphatidylcholine membranes were investigated as a function of hydrophobic mismatch by CD and oriented proton-decoupled (15)N solid-state NMR spectroscopies. In addition, the macroscopic phase and the orientational order of the phospholipid headgroups was analyzed by proton-decoupled (31)P NMR spectroscopy. Both, variations in the composition of the polypeptide (10-30 hydrophobic residues) as well as the fatty acid acyl chain of the phospholipid (10-22 carbons) were studied. At lipid-to-peptide ratios of 50, the peptides adopt helical conformations and bilayer macroscopic phases are predominant. The peptide and lipid maintain much of their orientational order even when the peptide is calculated to be 3 A too short or 14 A too long to fit into the pure lipid bilayer. A continuous decrease in the (15)N chemical shift obtained from transmembrane peptides in oriented membranes suggests an increasing helical tilt angle when the membrane thickness is reduced. This response is, however, insufficient to account for the full hydrophobic mismatch. When the helix is much too long to span the membrane, both the lipid and the peptide order are perturbed, an indication of changes in the macroscopic properties of the membrane. In contrast, sequences that are much too short show little effect on the phospholipid headgroup order, but the peptides exhibit a wide range of orientational distributions predominantly close to parallel to the membrane surface. A thermodynamic formalism is applied to describe the two-state equilibrium between in-plane and transmembrane peptide orientations.

Solid‐state nmr spectroscopy provides a robust method for investigating polypeptides that have been prepared by chemical synthesis and that are immobilized by strong interactions with solid surfaces or large macroscopic complexes. Solid‐state nmr spectroscopy has been widely used to investigate membrane polypeptides or peptide aggregates such as amyloid fibrils. Whereas magic angle spinning solid‐state nmr spectroscopy allows one to measure distances and dihedral angles with high accuracy, static membrane samples that are aligned with respect to the magnetic field direction allow one to determine the secondary structure of bound polypeptides and their orientation with respect to the bilayer normal. Peptide dynamics and the effect of polypeptides on the macroscopic phase preference of phospholipid membranes have been investigated in nonoriented samples. Investigations of the structure and topology of membrane channels, peptide antibiotics, signal sequences as well as model systems that allow one to dissect the interaction contributions in phospholipid membranes will be presented in greater detail. © 1999 John Wiley & Sons, Inc. Biopoly 51: 174–190, 1999

An approach is presented to selectively label the methionines of the colicin E1 and B channel domains, each about 200 residues in size, and use them for oriented solid-state NMR investigations. By combining site-directed mutagenesis, bacterial overexpression in a methionine auxotroph E. coli strain and biochemical purification, quantitative amounts of the proteins for NMR structural investigations were obtained. The proteins were selectively labeled with (15)N at only one, or at a few, selected sites. Multidimensional heteronuclear correlation high-resolution NMR spectroscopy and mass spectrometry were used to monitor the quality of isotopic labeling. Thereafter the proteins were reconstituted into oriented phospholipid bilayers and investigated by proton-decoupled (15)N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single (15)N methionine within its hydrophobic helix 9 region exhibited (15)N resonances that are characteristic of helices that are oriented predominantly parallel to the membrane surface at low temperature, and a variety of alignments and conformations at room temperature. This suggests that the protein can adopt both umbrella and pen-knife conformations.

The coat proteins of filamentous phage are first synthesized as transmembrane proteins and then assembled onto the extruding viral particles. We investigated the transmembrane conformation of the Pseudomonas aeruginosa Pf3 phage coat protein using proton‐decoupled 15N and 31P solid‐state NMR spectroscopy. The protein was either biochemically purified and uniformly labelled with 15N or synthesized chemically and labelled at specific sites. The proteins were then reconstituted into oriented phospholipid bilayers and the resulting samples analysed. The data suggest a model in which the protein adopts a tilted helix with an angle of ≈ 30° and an N‐terminal ‘swinging arm’ at the membrane surface.