Abstract
1,6-Diphenyl-1,3,5-hexatriene (DPH) is one of the most commonly used fluorescent probes to study dynamical and structural properties of lipid bilayers and cellular membranes via measuring steady-state or time-resolved fluorescence anisotropy. In this study, we present a limitation in the use of DPH to predict the order of lipid acyl chains when the lipid bilayer is doped with itraconazole (ITZ), an antifungal drug. Our steady-state fluorescence anisotropy measurements showed a significant decrease in fluorescence anisotropy of DPH embedded in the ITZ-containing membrane, suggesting a substantial increase in membrane fluidity, which indirectly indicates a decrease in the order of the hydrocarbon chains. This result or its interpretation is in disagreement with the fluorescence recovery after photobleaching measurements and molecular dynamics (MD) simulation data. The results of these experiments and calculations indicate an increase in the hydrocarbon chain order. The MD simulations of the bilayer containing both ITZ and DPH provide explanations for these observations. Apparently, in the presence of the drug, the DPH molecules are pushed deeper into the hydrophobic membrane core below the lipid double bonds, and the probe predominately adopts the orientation of the ITZ molecules that is parallel to the membrane surface, instead of orienting parallel to the lipid acyl chains. For this reason, DPH anisotropy provides information related to the less ordered central region of the membrane rather than reporting the properties of the upper segments of the lipid acyl chains.
Highlights
Free fluorescent probes and labels are commonly used in the studies of biological systems at both cellular and molecular levels
We studied the effect of the ITZ mole fraction (XITZ) on fluorescence anisotropy (r) of DPH embedded into the POPC/ITZ membrane of SUVs
The DPH fluorescence anisotropy can be reduced due to rotational diffusion, which is a function of the environment microviscosity
Summary
Free fluorescent probes and labels are commonly used in the studies of biological systems at both cellular and molecular levels. Fluorescent probes are very useful to monitor the lipid tail order, hydration at the membrane-water interface, membrane electrostatic properties, and dynamics of membrane components, such as diffusion or relaxation processes (Demchenko et al, 2009; Klymchenko and Kreder, 2014). Molecular probes or labels are unnatural elements of the studied systems. Molecular dynamics (MD) simulations have been used to understand the behavior of fluorescent probes, thereby facilitating the interpretation of experimental data and providing detailed information on the location and orientation of the probes, and their impact on surrounding lipid molecules (Faller, 2016; Kepczynski and Róg, 2016). E.g., NBD-labeled lipids (Filipe et al, 2014), F2N12S (Timr et al, 2015), and ATTO647N, ATTO532, KK114 (Mobarak et al, 2018)
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