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Selectively deuterated transmembrane peptides comprising alternating leucine-alanine subunits were examined in fluid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy in an effort to gain insight into the behavior of membrane proteins. Two groups of peptides were studied: 21-mers having a 17-amino-acid hydrophobic domain calculated to be close in length to the hydrophobic thickness of 1-palmitoyl-2-oleoyl phosphatidylcholine and 26-mers having a 22-amino-acid hydrophobic domain calculated to exceed the membrane hydrophobic thickness. (2)H NMR spectral features similar to ones observed for transmembrane peptides from single-span receptors of higher animal cells were identified which apparently correspond to effectively monomeric peptide. Spectral observations suggested significant distortion of the transmembrane alpha-helix, and/or potential for restriction of rotation about the tilted helix long axis for even simple peptides. Quadrupole splittings arising from the 26-mer were consistent with greater peptide "tilt" than were those of the analogous 21-mer. Quadrupole splittings associated with monomeric peptide were relatively insensitive to concentration and temperature over the range studied, indicating stable average conformations, and a well-ordered rotation axis. At high peptide concentration (6 mol% relative to phospholipid) it appeared that the peptide predicted to be longer than the membrane thickness had a particular tendency toward reversible peptide-peptide interactions occurring on a timescale comparable with or faster than approximately 10(-5) s. This interaction may be direct or lipid-mediated and was manifest as line broadening. Peptide rotational diffusion rates within the membrane, calculated from quadrupolar relaxation times, T(2e), were consistent with such interactions. In the case of the peptide predicted to be equal to the membrane thickness, at low peptide concentration spectral lineshape indicated the additional presence of a population of peptide having rotational motion that was restricted on a timescale of 10(-5) s.

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The orientation and motion of a model lysine-terminated transmembrane polypeptide were investigated by molecular dynamics simulation. Recent 2H NMR studies of synthetic polypeptides with deuterated alanine side chains suggest that such transmembrane polypeptides undergo fast, axially symmetric reorientation about the bilayer normal but have a preferred average azimuthal orientation about the helix axis. In this work, interactions that might contribute to this behavior were investigated in a simulated system consisting of 64 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and one alpha-helical polypeptide with the sequence acetyl-KK-(LA)11-KK-amide. In one simulation, initiated with the peptide oriented along the bilayer normal, the system was allowed to evolve for 8.5 ns at 1 atm of pressure and a temperature of 55 degrees C. A second simulation was initiated with the peptide orientation chosen to match a set of experimentally observed alanine methyl deuteron quadrupole splittings and allowed to proceed for 10 ns. Simulated alanine methyl group orientations were found to be inequivalent, a result that is consistent with 2H NMR observations of specifically labeled polypeptides in POPC bilayers. Helix tilt varied substantially over the durations of both simulations. In the first simulation, the peptide tended toward an orientation about the helix axis similar to that suggested by experiment. In the second simulation, orientation about the helix axis tended to return to this value after an excursion. These results provide some insight into how interactions at the bilayer surface can constrain reorientation about the helix axis while accommodating large changes in helix tilt.

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