Multiple Locations of Peptides in the Hydrocarbon
Core of Gel-Phase Membranes Revealed by Peptide <sup>13</sup>C to
Lipid <sup>2</sup>H Rotational-Echo Double-Resonance Solid-State Nuclear
Magnetic Resonance
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Abstract
Membrane
locations of peptides and proteins are often critical
to their functions. Solid-state rotational-echo double-resonance (REDOR)
nuclear magnetic resonance is applied to probe the locations of two
peptides via peptide <sup>13</sup>CO to lipid <sup>2</sup>H distance
measurements. The peptides are KALP, an α-helical membrane-spanning
peptide, and HFP, the β-sheet N-terminal fusion peptide of the
HIV gp41 fusion protein that plays an important role in HIV–host
cell membrane fusion. Both peptides are shown to have at least two
distinct locations within the hydrocarbon core of gel-phase membranes.
The multiple locations are attributed to snorkeling of lysine side
chains for KALP and to the distribution of antiparallel β-sheet
registries for HFP. The relative population of each location is also
quantitated. To the best of our knowledge, this is the first clear
experimental support of multiple peptide locations within the membrane
hydrocarbon core. These data are for gel-phase membranes, but the
approach should work for liquid-ordered membranes containing cholesterol
and may be applicable to liquid-disordered membranes with appropriate
additional analysis to take into account protein and lipid motion.
This paper also describes the methodological development of <sup>13</sup>CO–<sup>2</sup>H REDOR using the lyophilized I4 peptide that
is α-helical and <sup>13</sup>CO-labeled at A9 and <sup>2</sup>H<sub>α</sub>-labeled at A8. The I4 spins are well-approximated
as an ensemble of isolated <sup>13</sup>CO–<sup>2</sup>H spin
pairs each separated by 5.0 Å with a 37 Hz dipolar coupling.
A pulse sequence with rectangular 100 kHz <sup>2</sup>H π pulses
results in rapid and extensive buildup of REDOR (Δ<i>S</i>/<i>S</i><sub>0</sub>) with a dephasing time (τ).
The buildup is well-fit by a simple exponential function with a rate
of 24 Hz and an extent close to 1. These parameter values reflect
nonradiative transitions between the <sup>2</sup>H spin states during
the dephasing period. Each spin pair spends approximately two-thirds
of its time in the <sup>13</sup>CO–<sup>2</sup>H (<i>m</i> = ±1) states and approximately one-third of its time in the <sup>13</sup>CO–<sup>2</sup>H (<i>m</i> = 0) state and
contributes to the Δ<i>S</i>/<i>S</i><sub>0</sub> buildup during the former but not the latter time segments