Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase-dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance (EPR) spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that for DPPC membrane whose gel-to-fluid lipid phase transition occurs at 314 K, TEMPO is stabilized above (below) the bilayer interface if the membrane is in the gel (fluid) phase. For bilayers made of unsaturated lipids, DOPC and POPC, which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.