PHYSICAL STATE OF BULK AND PROTEIN-ASSOCIATED LIPID IN NICOTINIC ACETYLCHOLINE RECEPTOR-RICH MEMBRANE STUDIED BY LAURDAN GENERALIZED POLARIZATION AND FLUORESCENCE ENERGY-TRANSFER
Ss. Antollini et al., PHYSICAL STATE OF BULK AND PROTEIN-ASSOCIATED LIPID IN NICOTINIC ACETYLCHOLINE RECEPTOR-RICH MEMBRANE STUDIED BY LAURDAN GENERALIZED POLARIZATION AND FLUORESCENCE ENERGY-TRANSFER, Biophysical journal, 70(3), 1996, pp. 1275-1284
The spectral properties of the fluorescent probe laurdan (6-dodecanoyl
-2-dimethylaminonaphthalene were exploited to learn about the physical
state of the lipids in the nicotinic acety]choline receptor (AChR)-ri
ch membrane and compare them with those in reconstituted liposomes pre
pared from lipids extracted from the native membrane and those formed
with synthetic phosphatidylcholines. In all cases redshifts of 50 to 6
0 nm were observed as a function of temperature in the spectral emissi
on maximum of laurdan embedded in these membranes. The so-called gener
alized polarization of laurdan exhibited high values (0.6 at 5 degrees
C) in AChR-rich membranes, diminishing by similar to 85% as temperatu
re increased, but no phase transitions with a clear T-m were observed.
A still unexploited property of laurdan, namely its ability to act as
a fluorescence energy transfer acceptor from tryptophan emission, has
been used to measure properties of the protein-vicinal lipid. Energy
transfer from the protein in the AChR-rich membrane to laurdan molecul
es could be observed upon excitation at 290 nm. The efficiency of this
process was similar to 55% for 1 mu M laurdan. A minimum donor-accept
or distance r of 14 +/- 1 Angstrom could be calculated considering a d
istance 0 < H < 10 Angstrom for the separation of the planes containin
g donor and acceptor molecules, respectively, This value of r correspo
nds closely to the diameter of the first-shell protein-associated lipi
d, A value of similar to 1 was calculated for K-r, the apparent dissoc
iation constant of laurdan, indicating no preferential affinity for th
e protein-associated probe, i.e., random distribution in the membrane.
From the spectral characteristics of laurdan in the native AChR-rich
membrane, differences in the structural and dynamic properties of wate
r penetration in the protein-vicinal and bulk bilayer lipid regions ca
n be deduced. We conclude that 1) the physical state of the bulk lipid
in the native AChR-rich membrane is similar to that of the total lipi
ds reconstituted in liposomes, exhibiting a decreasing polarity and an
increased solvent dipolar relaxation at the hydrophilic/hydrophobic i
nterface upon increasing the temperature; 2) the wavelength dependence
of laurdan generalized polarization spectra indicates the presence of
a single, ordered (from the point of view of molecular axis rotation)
-liquid (from the point of view of lateral diffusion) lipid phase in t
he native AChR membrane; 3) laurdan molecules within energy transfer d
istance of the protein sense protein-associated lipid, which differs s
tructurally and dynamically from the bulk bilayer lipid in terms of po
larity and molecular motion and is associated with a lower degree of w
ater penetration.