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 n–th 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 n–th 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 Å 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.

Effects of Spin-Labels on Membrane Burial Depth of MARCKS-ED Residues

“…For positions Lys 3 and Lys 5 , the native residues are closer to the experimental results, and for positions Lys 14 and Lys 24 , the spin labels and native residues are equally close to the experimental depths. The difference between the simulation depth and the experimental depth could arise from the HMMM membrane model or the uncertainty in the experimental values (12). Phe 13 -SL is the only spin label that has a comparable depth to that of the native residue, which is in agreement with the experimental measurements by Ellena et al (13).…”

Molecular modeling of lipid probes and their influence on the membrane

“…Spin-labeled lipids are commonly used as fluorescence quenchers in depth-dependent quenching where they are added in addition to fluorophores, i.e. one has two different probes where one inhibits the other [90,91]. Here one of the attached chemical groups have an unpaired spin typically from a stabilized radical, these are the same types of molecules used as in EPR labeling experiments (see below).…”

“…To probe different depths within the bilayer five different Doxyl-labeled lipids have been investigated, where the spin-label moiety was covalently bound to different carbon atoms (positions n = 5, 7, 10, 12, and 14) of the sn-2 chain of the phospholipid [90]. The spin probes turn out to be broadly distributed across the membrane with heterogeneous neighborhoods but in general the labeled carbons are at similar depths as their unlabeled counterparts at the same chain position.…”

“…A broader and more heterogeneous distribution was found for a headgroup-attached Tempo spin label which due to its hydrophobic nature was deeper in the membrane than unlabeled headgroups. Depending on the concentration of Tempo-labeled lipids, the depth of the Tempo moiety was around 14 to 18Å from the membrane center [90]. Comparison of the MDestimated depths with the experimental suggestions allow to determine potential sources of error in depth-dependent fluorescence quenching studies [90].…”

Chapter 2. Computational approaches for simulating motional EPR spectra

“…110 Other recent developments include ff for phospholipid spin probes for atomistic simulations in lipid bilayers. 111 For all atom (AA) simulations Hustedt and co-workers parameterised MTSL ffs consistent with AMBER99 ff employed in AMBER suite. 68 A set of ff parameters has been developed for both 5CB/8CB nematics and cholesteric spin probe for AA MD simulations in both AMBER 97 and GRO-MACS.…”