Measuring Raft Size as a Function of Membrane Composition in PC-Based Systems:  Part 1 − Binary Systems

Abstract

This work applied two steady-state fluorescence techniques to detect nanoscopic membrane domains in a binary dimyristoylphosphocholine (DMPC)-cholesterol system and a ternary dioleoylphosphocholine (DOPC)-dipalmitoylphosphocholine (DPPC)-cholesterol system. A polarity-induced spectral shift in the emission spectra of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) in combination with a Förster resonance energy transfer (FRET) assay agreed with the phase diagrams that have been published for these systems and were observed to be useful tools in the detection of membrane heterogeneities. The DAN-PC/dehydroergosterol (DHE) FRET pair was found to be best suited for use with these steady-state techniques because of their differential partitioning between phases, although a high acceptor concentration was needed to obtain accurate measurements. In the binary system, this high probe concentration was found to be perturbing, but in more representative ternary systems, the high probe concentration no longer disrupted the phase behavior of the system. This FRET pair allowed for the calculation of nanometer-scale domain sizes in model ternary systems, using the two steady-state fluorescence techniques along with a clear and straightforward model.

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

“…At room temperature, a membrane composed of DMPC and 0% cholesterol exists in a non-raft-like (liquid-disordered) phase, whereas at 40 and 60% cholesterol, the membrane exists in a raft-like (liquid-ordered phase) (27,28). In vitro, the lipid composition of lipid rafts is often ϳ30% (74 -77).…”

“…The liposomes were composed of DMPC with 0, 20, 40, or 60% cholesterol (or ergosterol). At room temperature, DMPC with 0% cholesterol exists in a nonraft-like state (liquid disordered); at 40 and 60% cholesterol, DMPC exists in a raft-like state (liquid ordered); and at 20% cholesterol, both phases coexist (27,28).…”

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution

“…One of the most promising direct methodologies for studying raft domains is Förster resonance energy transfer (FRET), which has a nanometer scale resolution ( 54,55 ). Here we complemented the results obtained using Triton X-100 with FRET measurements between the single-tryptophan intrinsic fl uorescence of the ␥ M4 peptide or the 51 tryptophans of the whole AChR acting as donor, and the fl uorescent probes DHE or Laurdan as acceptors.…”

Planar Lipid Bilayers Containing Gramicidin A as a Molecular Sensing System Based on an Integrated Current

“…The appearance of the two conductance levels in the DOPE-rich bilayer implies that the bilayer is not homogenous. The presence of lipid rafts in vesicles of a DOPC-DPPC-Chol system has been reproted by using fluorometric 36,37 and small-angle neutron scattering techniques. 38 We suppose that there exist lipid rafts in the DPPC-DOPE bilayer.…”