Depth-dependent solvent relaxation in membranes: wavelength-selective fluorescence as a membrane dipstick

Membrane penetration depth represents an important parameter which can be used to define the conformation and topology of membrane proteins and probes. We have previously characterized a set of fluorescence spectroscopic approaches, collectively referred to as wavelength-selective fluorescence, as a powerful tool to monitor microenvironments in the vicinity of reporter fluorophores embedded in the membrane. Since several membrane parameters that characterize local environments such as polarity, fluidity, segmental motion, degree of water penetration, and the ability to form hydrogen bonds are known to vary as a function of depth of penetration into the membrane, we propose that wavelength-selective fluorescence could provide a novel approach to investigate the depth of membrane penetration of a reporter fluorophore. We test this hypothesis by demonstrating that chemically identical fluorophores, varying solely in terms of their localization at different depths in the membrane, experience very different local environments, as judged by wavelength-selective fluorescence parameters. We used two anthryoloxy stearic acid derivatives where the anthroyloxy group has previously been found to be either shallow (2-AS) or deep (12-AS). Our results show that the anthroyloxy moiety of 2- and 12-AS experiences different local membrane microenvironments, as reflected by varying extents of red-edge excitation shift (REES) as well as varying degrees of wavelength dependence of fluorescence polarization and lifetime and rotational correlation times. We attribute these results to differential rates of solvent reorientation in the immediate vicinity of the anthroyloxy group as a function of its membrane penetration depth. We thus provide evidence, for the first time, of depth-dependent solvent relaxation which can be used as a membrane dipstick