Depth-Dependent Solvent Relaxation in Membranes:  Wavelength-Selective Fluorescence as a Membrane Dipstick

Abstract

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.

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

“…While the center of the bilayer is nearly isotropic, the upper portion, only a few angstroms away toward the membrane surface, is highly ordered. 38,[101][102][103][104][105] Properties such as polarity, fluidity, segmental motion, ability to form hydrogen bonds and extent of solvent penetration vary in a depth-dependent manner in the membrane. The interfacial region in membranes is the most important region so far as the dynamics and function of the membrane is concerned.…”

“…38,112 It is therefore the membrane interface which is most likely to display REES. 105,[113][114][115] The application of REES to study the organization and dynamics of membranes has been reviewed recently. …”

“…Such extrinsic fluorescent probes are widely used to study the dynamics of proteins and membranes. 74,105,113,114,176 The advantage of this approach is that one has a choice of the fluorescent label to be used and, therefore, specific probes with appropriate characteristics can be designed for specific applications. Many of these probes display high sensitivity to the polarity of the local environment.…”

Modulation of Fluorophore Environment in Host Membranes of Varying Charge

“…1(c) shows the chemical structure of 2-AS, the fluorescent probe used. Fatty acids labeled with spectroscopic reporter groups have proved to be useful membrane probes [16]. The anthroyloxy fatty acids such as 2-AS in which an anthracene group is attached by an ester linkage to an alkyl chain have been extensively used as fluorescent probes in membranes [16,[32][33][34][35].…”

“…This makes REES and related techniques extremely useful in biology since hydration plays a crucial modulatory role in a large number of important cellular events [7,8] such as lipid-protein interactions [9] and ion transport [10][11][12]. We have earlier shown that REES and related techniques (wavelength-selective fluorescence 459 approach) serve as a powerful tool to monitor organization and dynamics of membrane-bound probes and peptides [1,[13][14][15][16][17].…”