Peptides F5GWALP23, F4,5GWALP23, Y4GWALP23, and W18GWALP23 (Table 1 ) were synthesized on a model 433A synthesizer from
Applied Biosystems
by Life Technologies (Foster City, CA) using solid-phase methods,
as described previously.17 (link) Typically, two
deuterated alanines of differing isotope abundances were incorporated
into each synthesized peptide. Peptides were purified as described19 (link),21 (link) using an octyl silica column (Zorbax Rx-C8, 9.4 × 250 mm, 5
μm particle size; Agilent Technologies, Santa Clara, CA) and
a gradient of 97–100% methanol (with 0.1% trifluoroacetic acid)
over 28 min. Final peptide purity (>97%) was confirmed by reversed-phase
HPLC, and peptide identity, by mass spectrometry (Figure S1 of theSupporting Information ).
Solid-state 2H NMR experiments,
using mechanically aligned
samples, were performed using methods that have been described previously.17 (link) Mechanically aligned samples (1:60, peptide/lipid;
45% hydration, w/w) were prepared using DOPC, DMPC, or DLPC lipid
from Avanti Polar Lipids (Alabaster, AL) and deuterium-depleted water
from Cambridge Isotope Laboratories (Andover, MA). Bilayer alignment
within each sample was confirmed using 31P NMR at 50 °C
on a Bruker (Billerica, MA) Avance 300 spectrometer. Deuterium NMR
spectra were recorded at 50 °C using both β = 0° (bilayer
normal parallel to magnetic field) and β = 90° macroscopic
sample orientations on a Bruker Avance 300 spectrometer utilizing
a quadrupolar echo pulse sequence22 with
90 ms recycle delay, 3.2 μs pulse length, and 115 μs echo
delay. Between 0.6 and 1.5 million scans were accumulated during each 2H NMR experiment. An exponential weighting function with 100
Hz line broadening was applied prior to Fourier transformation.
Helix orientations were analyzed by means of a semistatic “GALA”
method based on three adjustable parameters: the average tilt τo of the helix axis, the average azimuthal rotation ρo about the helix axis, and a principal order parameter Szz, as described.7 (link),17 (link) An additional
three-parameter modified Gaussian method is available, based on τo, ρo, a distribution width σρ,
and a fixed στ.16 (link) We also employed
this modified Gaussian method, but στ was fixed at either
15° (DLPC) or 9° (DOPC; seeDiscussion ) instead of the previously assumed value of 0° for στ.
For the analysis of helix rotation, we analyzed some pairwise residue
separation distances using a recently described procedure.23 (link) Distances were compared to hydrophobic thicknesses
of 20.9 Å for DLPC24 (link) and 27.2 Å
for DOPC,25 which are based on the location DC of the Gibbs dividing surface for the hydrocarbon
region of the bilayer.25
Applied Biosystems
by Life Technologies (Foster City, CA) using solid-phase methods,
as described previously.17 (link) Typically, two
deuterated alanines of differing isotope abundances were incorporated
into each synthesized peptide. Peptides were purified as described19 (link),21 (link) using an octyl silica column (Zorbax Rx-C8, 9.4 × 250 mm, 5
μm particle size; Agilent Technologies, Santa Clara, CA) and
a gradient of 97–100% methanol (with 0.1% trifluoroacetic acid)
over 28 min. Final peptide purity (>97%) was confirmed by reversed-phase
HPLC, and peptide identity, by mass spectrometry (Figure S1 of the
Solid-state 2H NMR experiments,
using mechanically aligned
samples, were performed using methods that have been described previously.17 (link) Mechanically aligned samples (1:60, peptide/lipid;
45% hydration, w/w) were prepared using DOPC, DMPC, or DLPC lipid
from Avanti Polar Lipids (Alabaster, AL) and deuterium-depleted water
from Cambridge Isotope Laboratories (Andover, MA). Bilayer alignment
within each sample was confirmed using 31P NMR at 50 °C
on a Bruker (Billerica, MA) Avance 300 spectrometer. Deuterium NMR
spectra were recorded at 50 °C using both β = 0° (bilayer
normal parallel to magnetic field) and β = 90° macroscopic
sample orientations on a Bruker Avance 300 spectrometer utilizing
a quadrupolar echo pulse sequence22 with
90 ms recycle delay, 3.2 μs pulse length, and 115 μs echo
delay. Between 0.6 and 1.5 million scans were accumulated during each 2H NMR experiment. An exponential weighting function with 100
Hz line broadening was applied prior to Fourier transformation.
Helix orientations were analyzed by means of a semistatic “GALA”
method based on three adjustable parameters: the average tilt τo of the helix axis, the average azimuthal rotation ρo about the helix axis, and a principal order parameter Szz, as described.7 (link),17 (link) An additional
three-parameter modified Gaussian method is available, based on τo, ρo, a distribution width σρ,
and a fixed στ.16 (link) We also employed
this modified Gaussian method, but στ was fixed at either
15° (DLPC) or 9° (DOPC; see
For the analysis of helix rotation, we analyzed some pairwise residue
separation distances using a recently described procedure.23 (link) Distances were compared to hydrophobic thicknesses
of 20.9 Å for DLPC24 (link) and 27.2 Å
for DOPC,25 which are based on the location DC of the Gibbs dividing surface for the hydrocarbon
region of the bilayer.25
