Fluorescence investigation of laterally phase-separated cholesterol rich domains in model lipid membranes using the membrane probe 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine (A)

Abstract

This work utilizes the fluorescence properties of the membrane probes 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine (DANSYL), and ergosta-5,7,9(11),22-tetraen-3β-ol (ERGO) to detect and characterize laterally phase-separated, cholesterol-rich domains in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol, and egg lecithin(Egg-PC)/cholesterol model membrane systems. Specifically, the DANSYL membrane probe shows evidence of lateral phase separations in DMPC/Cholesterol and Egg-PC/Cholesterol membrane mixtures and can reveal liquid-ordered to liquid-disordered phase boundaries. Fluorescence resonance energy transfer from ERGO to DANSYL is used to characterize the size of the putative laterally phase separated cholesterol-rich domains in model membrane systems. The DANSYL red shifting data provides evidence for the presence of a lateral phase separation in the cholesterol regime that was thought to be only in the liquid ordered phase. Simulations were conducted using two vertically-coupled square two-dimensional lattices to simulate membrane bilayers containing a uniform size distribution of cholesterol immiscible domains of a predetermined size distribution. We substitute cholesterols and phospholipids with their fluorescent analogs and calculate the efficiency of energy transfer as a function of acceptor concentration for four membrane configurations. We show that the FRET model yields good size estimates for domains that range between 1 and 25 nm. We also find that the assumed fluorophore configuration in the FRET model leads to a constant under-prediction of these values. Finally, we demonstrate that when two parameters are open to the fit, the FRET model adequately predicts the donor partition coefficient in addition to the domain size. Finally, we present a steady-state and time-resolved fluorescence emission spectra analysis of the membrane probe 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine (DANSYL) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine and cholesterol multi-lamellar vesicles prepared by modified rapid solvent exchange. We report that the dose-dependent cholesterol-induced blue shifts in the steady-state fluorescence emission spectra in 1,2-dimyristoyl-sn-glycero-3-phosphocholine multi-lamellar vesicles are due to complex solvent effects that include time-dependent dipolar relaxation, and the formation of internal charge transfer states. A key finding of this study is identification of two distinguishable DANSYL populations existing at both shallow and deep locations in membrane, these two DANSYL populations are evidence of the presence of laterally phase separated domains.%%%%Ph.D., Chemical Engineering – Drexel University, 2004