Time-Dependent Stokes Shifts of Fluorescent Dyes in the Hydrophobic Backbone Region of a Phospholipid Bilayer:  Combination of Fluorescence Spectroscopy and Ab Initio Calculations

Abstract

We explored the time-dependent Stokes shifts of fluorescent dyes containing an anthroyloxy chromophore (2-AS, 9-AS, and 16-AP) in bilayers composed of palmitoyl-oleoyl-phosphatidylcholine. The obtained data revealed a nontrivial solvation response of these dyes, which are located in the backbone region of the bilayer with a gradually increasing depth. For comparison, steady-state emission spectra in the neat solvents of various polarities and viscosities were also recorded. The results indicate that on the short picosecond time scale the AS dyes undergo complex photophysics including formation of states with a charge-transfer character. This observation is supported by ab initio calculations of the excited states of 9-methylanthroate. The slower nanosecond part of the relaxation process can be attributed to the solvation response of the dyes. A slowdown in solvent relaxation is observed upon moving toward the center of the bilayer. A mechanism similar to preferential solvation present in the mixture of a polar and nonpolar solvent is considered to explain the obtained data.

Profiling of dynamics in protein–lipid–water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths

“…This indicates that fewer water molecules are available for relaxing these states, which is a measure for the local water concentration and related polarity. This finding is in agreement with fluorescence data obtained with fluorescent dye in phospholipid systems, located at various depths in the bilayer (Hutterer et al 1996; Jurkiewicz et al 2006; Sýkora et al 2007). On the other hand, the label dynamics slows down on entering the headgroup region of the membrane (increase of τ LD1,2 ).…”

“…Hydration water in the hydrophobic interior of membranes leads to polarity and hydration profiles across it, which are fundamental in maintaining the membrane’s architecture as well as in transport and insertion processes (Epand and Kraayenhof 1999; Klymchenko et al 2004; Sýkora et al 2007). This work is based on solvent relaxation (Jurkiewicz et al 2006; Sýkora et al 2007), i.e., the finding that the excited state of fluorescent probes is sensitive to the ability of the surrounding water molecules to dielectrically stabilize the ground and excited state dipoles of the probe (Lakowicz 2006). In addition, hydration-water dynamics is an important factor for the lipid dynamics and the dynamics of the membrane-embedded proteins (Wood et al 2007).…”

Oxidized Phosphatidylcholines Facilitate Phospholipid Flip-Flop in Liposomes

“…The obtained values for t in POPC were as follows: Dtmac 1.17, Laurdan 3.12, and Patman 4.15 ns. Because the time-dependent red shifts for 9-AS and 16-AP involve not only SR but also intramolecular relaxation (42), an interpretation of the observed kinetics for these two probes in terms of their environment mobility would be ambiguous and was excluded from analysis. Changes in the total spectral shift (Dn), however, are informative, as both contributions (i.e., from SR and from intramolecular relaxation) affect Dn in the same way, i.e., increasing the spectral shift when the polarity of the environment increases.…”

Electrochemical Measurements on Supported Phospholipid Bilayers: Preparation, Properties and Ion Transport Using Incorporated Ionophores

“…They are based on many principles, e.g., passive diffusion, facilitated diffusion, ion pumps and channels (e.g., in cases of Ca 2þ , K þ , Na þ ), endocytosis and exocytosis (e.g., in cases of larger objects and particles, such as bacteria, viruses) [6]. Various techniques have been applied to the study of the membrane formation and of the transporting processes, e.g., fluorescence microscopy [7], fluorescence lifetime correlation spectroscopy combined with lifetime tuning [8], combination of fluorescence spectroscopy and ab initio calculations [9], solvent relaxation technique [10], or confocal fluorescence correlation spectroscopy [11]. For elucidation of the basic principles dealing with the transport of particles across the membranes, the investigation of liquid-liquid systems, i.e., systems of two immiscible liquids separated by a phospholipid layer, can be useful (e.g., [12 -15]).…”