Molecular dynamics simulations of depth distribution of spin-labeled phospholipids within lipid bilayer

Abstract

Spin-labeled lipids are commonly used as fluorescence quenchers in studies of membrane penetration of dye-labeled proteins and peptides using depth-dependent quenching. Accurate calculations of depth of the fluorophore rely on the use of several spin labels placed in the membrane at various positions. The depth of the quenchers (spin probes) has to be determined independently; however, experimental determination of transverse distributions of spin probe depths is difficult. In this Article, we use molecular dynamics (MD) simulations to study the membrane behavior and depth distributions of spin-labeled phospholipids in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. To probe different depths within the bilayer, a series containing five Doxyl-labeled lipids (n-Doxyl PC) has been studied, in which a spin moiety was covalently attached to nth carbon atoms (where n = 5, 7, 10, 12, and 14) of the sn-2 stearoyl chain of the host phospholipid. Our results demonstrate that the chain-attached spin labels are broadly distributed across the model membrane and their environment is characterized by a high degree of mobility and structural heterogeneity. Despite the high thermal disorder, the depth distributions of the Doxyl labels were found to correlate well with their attachment positions, indicating that the distribution of the spin label within the model membrane is dictated by the depth of the nth lipid carbon atom and not by intrinsic properties of the label. In contrast, a much broader and heterogeneous distribution was observed for a headgroup-attached Tempo spin label of Tempo-PC lipids. MD simulations reveal that, due to the hydrophobic nature, a Tempo moiety favors partitioning from the headgroup region deeper into the membrane. Depending on the concentration of Tempo-PC lipids, the probable depth of the Tempo moiety could span a range from 14.4 to 18.2 Å from the membrane center. Comparison of the MD-estimated immersion depths of Tempo and n-Doxyl labels with their suggested experimental depth positions allows us to review critically the possible sources of error in depth-dependent fluorescence quenching studies.

Figure 1. Distribution of n–Doxyl–PCs

Snapshots of a lipid bilayer containing 12 n–Doxyl–PC and 116 POPC molecules (11 mol % of bilayer spin labeling) taken at the end of MD simulations (t=100 ns). The acyl chain–attached doxyl labels revealed the high degree of mobility and structural heterogeneity in the bilayer. The bilayer is shown as sticks in olive and n–Doxyl–PCs are color–coded. The Doxyl moieties are shown with van der Waals representation. Water molecules are not shown for clarity.

Figure 2. Distribution of 5–Doxyl–PC

(A) MD snapshots of lipid bilayers composed of 5–Doxyl–PC and POPC with a ratio of 34:94 (29 mol % of bilayer spin–labeling). The bilayer shown in stick representations are olive and the 5–Doxyl–PC lipids are brown. The Doxyl labels are shown using van der Waals representation. (B) Probability densities of 5–Doxyl label shown for two different spin label concentrations of 11 (black) and 29 (red) mol %, respectively. Probability density peaks calculated from the center of a POPC bilayer are also shown.

Figure 3. Spin Label Depths

Transverse mass density distributions of Tempo–PC and n–Doxyl–PCs in a POPC bilayer observed in MD simulations. The distributions of individual components are averaged over the last 50 ns of the MD sampling and superimposed on the profiles of a POPC bilayer (solid black) and water (dashed yellow). (A) Contributions from the spin label moieties are shown color–coded: Tempo (solid red); 5–D, 5–Doxyl (solid ochre); 7–D, 7–Doxyl (dashed cyan); 10–D, 10–Doxyl (dotted green); 12–D, 12–Doxyl (solid orange); 14–D, 14–Doxyl (solid blue). The spin label profiles were multiplied by a factor of 15 for clarity. (B) Contributions from individual components of the POPC lipids: phosphate groups, PO₄, (solid dark yellow) and carbon atoms of the sn–2 acyl chain C20, C22, C25, C27, C29 (see Scheme 1 for atom numbering). The profiles for the carbon atoms use the same coloring scheme as in panel A.

Figure 4. Distribution of Tempo–PC

MD snapshots of lipid bilayers composed of Tempo–PC and POPC with a ratio of 12:116 (top) and 32:96 (bottom) that correspond to 10.9 mol % and 28 mol % of bilayer spin–labeling, respectively. The bilayer shown in stick representations are olive and the Tempo-PC lipids are red. The Tempo moieties are shown using van der Waals representation.

Figure 5. Depth of Tempo–PC

Probability densities of the Tempo label are compared for two different concentrations of 11 (black) mol % and 28 (red) mol %, respectively. To account for different concentrations, the areas under mass density profiles were normalized to 1. Due to the asymmetry of the density distributions, the most probable depth of the spin label was calculated from center–of–mass of their probability profiles. The corresponding COM depths calculated from the center of a POPC bilayer were found to be 14.4 Å and 18.2 Å, respectively.

Figure 6. Reorientational Dynamics of Spin Labels

Rotation autocorrelation functions for the spin label group of Tempo–PC and n–Doxyl PC in a POPC bilayer. Rotational times τ_α were calculated from fitting C₂(t) to a two–exponential decay (solid lines) and averaging fast and slow components.

Figure 7. Comparison of Experimental and MD–Estimated Depths

The MD–estimated depths of 5, 7, 10, 12 and 14–Doxyl labels (squares) are plotted versus the lipid chain carbon atom number. The MD–depths of 7, 10, and 12–Doxyls demonstrate systematic upward deviations from the calibration line derived from the carbon atom depths (circles) in the unlabeled sn–2 lipid chain based on the X-ray/NMR data.^,,

Scheme 1. Molecular Structure of Spin–Labeled Lipids

Structure and atom numbering of unlabeled POPC, Tempo-PC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(TEMPO)choline), n–Doxyl–PC (1-palmitoyl-2-stearoyl-(n–Doxyl)/sn-glycero-3-phosphocholine) spin–labeled lipids. A Tempo label (in red) is covalently attached to a headgroup and a Doxyl moiety (color–coded) is introduced to a variety of positions down to the sn–2 stearoyl chain of the host lipid.