Disorder in Order-Related Membrane Biophysical Parameters: An In-Depth Analysis of Di-4-ANEPPDHQ Generalized Polarization

Molecular order-related bulk membrane properties that substantially modulate protein functions can be examined with environment-sensitive probes, such as the prototypical and most widely applied solvatochromic Laurdan, whose spectral parameters change depending on the local hydrophobicity. Di-4-ANEPPDHQ is a widely accepted Laurdan alternative with more favorable spectral properties suitable for standard imaging, and information provided by the two fluorophores is generally considered equivalent. In our study, using fluorescence-based experimental approaches, we demonstrate that different sterols distinctly alter di-4-ANEPPDHQ spectral properties, and these changes do not correlate with those observed with Laurdan. Our molecular dynamics simulations reveal that this may be caused by their distinct depth localization in bilayers since the sensor moiety of di-4-ANEPPDHQ is localized in the vicinity of the membrane-water interface as opposed to that of Laurdan lying near the hydrophobic core. Therefore, di-4-ANEPPDHQ can be considered as a complementary tool rather than an equivalent substitute of Laurdan.