A Novel Fluorescent Probe That Senses the Physical State of Lipid Bilayers

Abstract

Cell membrane lipids and proteins are heterogeneously distributed in the membrane plane. In recent years, much attention has been paid to the heterogeneous distribution of the lipid components, particularly the formation of cholesterol-rich domains that are thought to be important in signaling processes. This has led to renewed interest in the phase diagrams of complex lipid mixtures, such as three-component mixtures containing phospholipids and cholesterol. We report here a novel fluorescent probe (NBD-R595) that is useful for exploring the phase behaviors of one-, two-, and three-component large unilamellar vesicles. In one-component fluid-phase membranes, the probe has the expected spectral characteristic of monomeric 7-nitrobenzo-2-oxa-1,3-diazol, with a fluorescence maximum of 540 nm when excited at 470 nm. But below the gel-to-liquid crystalline phase transition temperature, an additional emission peak appears at approximately 610 nm, because of Förster resonance energy transfer from NBD-R595 monomers to NBD-R595 Jelley aggregates of limited size formed by the association of 7-nitrobenzo-2-oxa-1,3-diazol moieties. This may be the first report of Förster resonance energy transfer from a single fluorophore in two different physical states. In a test of the probe, we found NBD-R595 to be remarkably sensitive to the molar composition of large unilamellar vesicles formed from cholesterol, distearoylphosphatidylcholine, and dioleoylphosphatidylcholine.

Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO

“…Similar, but less extensive red shifts have been observed with a nitrobenzooxadiazol (NBD) dye in LUVs maintained above and below the gel-to-liquid crystalline phase transition temperature and has been attributed to FRET from dye monomers to dye aggregates of J-type formed within the bilayer. 51 This behavior is consistent with the appearance of the sharp absorption peak (Fig. 6a).…”

Mammalian sphingoid bases: Biophysical, physiological and pathological properties

“…Nonetheless, changes in pH from an acidic to a neutral environment alter the hydrogen-bonding network of SO aggregates, from intramolecular to intermolecular [99], and is therefore expected to influence membrane biophysical properties. Moreover, SO might affect membrane surface charge in a manner dependent on both membrane lipid composition and pH, suggesting that a local enrichment in SO would affect the electrostatic interactions of membrane proteins and consequently protein activity.…”

Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages

“…1C). When 0.1 µM of NBD-cholesterol was used, which represents a concentration reported in several previous studies (10,19,20), for up to 12 h of incubation, real-time imaging yielded dim fluorescent images. This finding was confirmed by quantitative FI detection, in which the FI of the cell lysate was undetectable following 12 h incubation.…”