Molecular dynamics simulations of DiI-C₁₈(3) in a DPPC lipid bilayer

We performed a 40 ns simulation of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-C₁₈(3)) in a 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) bilayer in order to facilitate interpretation of lipid dynamics and membrane structure from fluorescence lifetime, anisotropy, and fluorescence correlations spectroscopy (FCS). Incorporation of DiI of 1.6 to 3.2 mol% induced negligible changes in area per lipid but detectable increases in bilayer thickness, each of which are indicators of membrane structural perturbation. The DiI chromophore angle was 77 ± 17° with respect to the bilayer normal, consistent with rotational diffusion inferred from polarization studies. The DiI headgroup was located 0.63 nm below the lipid head group–water interface, a novel result in contrast to some popular cartoon representations of DiI but consistent with DiI’s increase in quantum yield when incorporated into lipid bilayers. Importantly, the fast component of rotational anisotropy matched published experimental results demonstrating that sufficient free volume exists at the sub-interfacial region to support fast rotations. Simulations with non-charged DiI head groups exhibited DiI flip-flop, demonstrating that the positively-charged chromophore stabilizes the orientation and location of DiI in a single monolayer. DiI induced detectable changes in interfacial properties of water ordering, electrostatic potential, and changes in P–N vector orientation of DPPC lipids. The diffusion coefficient of DiI (9.7 ± 0.02 × 10⁻⁸ cm² s⁻¹) was similar to the diffusion of DPPC molecules (10.7 ± 0.04 × 10⁻⁸ cm² s⁻¹), supporting the conclusion that DiI dynamics reflect lipid dynamics. These results provide the first atomistic level insight into DiI dynamics, results essential in elucidating lipid dynamics through single molecule fluorescence studies.