NMR, calorimetric, spin-label, and optical studies on a trifluoromethyl-substituted styryl molecular probe in dimyristoylphosphatidylcholine vesicles and multilamellar suspensions: a model for location of optical probes.
NMR, calorimetric, and optical spectroscopic studies have been performed on a trifluoromethyl-substituted styryl molecular probe bound to vesicles and multilamellar suspensions formed from dimyristoylphosphatidylcholine (DMPC). In the fluorine NMR spectrum at 35 degrees C there are two partially resolved resonances, but these collapse to an apparently single resonance at temperatures above 60 degrees C. However, a line-shape analysis is not consistent with exchange between two sites on an NMR time scale, and the two resonances are assumed to be due to probe sites in the inner and outer leaflets of the vesicles. Two fluorescence lifetimes, each associated with one of these sites, characterize the decay curves for the molecular probe bound to DMPC vesicles. The shift reagent Eu(FOD)3 and several nitroxide spin labels covalently bound to lipophilic structures strongly attenuate the lower frequency component of the fluorine NMR spectrum and also shift the other resonance to higher frequencies. The effect of two spin labels on the probe fluorine T2 relaxation time has been used to estimate the distance between the spin label unpaired electron and the trifluoromethyl group. The location of the spin label site in the membrane was determined from the effect of the unpaired electron on the lipid 13C linewidths. A model for the location of the probe in the bilayer was developed from the above information and refined using molecular mechanics calculations on a probe-DMPC lipid complex. The long axis of the probe parallels the bilayer normal; the styryl-group portion of the optical chromophore is located slightly below the glycerol backbone, and the remainder of the chromophore extends well into the hydrophobic region of the bilayer. Therefore, the optical properties of the probe should not be significantly influenced by alterations of the membrane surface charge density. Parameters derived from DSC studies in the gel-to-lipid crystal phase transition of DMPC are extremely sensitive to the probe. Even at 0.0001 mol fraction of probe, the transition is substantially broadened, and the delta H for the transition has increased, just as one predicts for the formation of a tight complex described above.
Bammel
B P
BP
Department of Chemistry, Georgia State University, Atlanta 30303-3053.
Hamilton
D D
DD
Haugland
R P
RP
Hopkins
H P
HP
Schuette
J
J
Szalecki
W
W
Smith
J C
JC
D002151:Calorimetry; D004134:Dimyristoylphosphatidylcholine; D004578:Electron Spin Resonance Spectroscopy; D005466:Fluorocarbons; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013343:Styrenes; D013696:Temperature; D013816:Thermodynamics
D005466:Fluorocarbons; D013113:Spin Labels; D013343:Styrenes; D004134:Dimyristoylphosphatidylcholine