Electron spin resonance and steady-state fluorescence polarization studies of lipid bilayers containing integral proteins

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Abstract
Equations are derived that describe changes in the steady-state fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) or in the spectrum of ESR nitroxide spin-labeled lipid probes as a function of the intrinsic molecule concentration in lipid bilayer membranes. The equations are independent of any membrane model. They are valid when a DPH probe or a spin-labeled chain is equivalent to an unlabeled lipid hydrocarbon chain only as far as their general space-filling properties are concerned. Cases where the bilayer is either in a single homogeneous phase or in a 2-phase region are considered. The equations are used to analyze ESR data from delipidated sarcoplasmic reticulum membranes and from egg yolk phosphatidylcholine bilayers containing Ca2+-ATPase, and DPH data from dipalmitoylphosphatidylcholine (DPPC) bilayers containing Ca2+-ATPase both for T > Tc [pure lipid main phase transition temperature]. The following conclusions were derived: Ca2+-ATPase oligomers are randomly distributed, for the concentrations studied, in the fluid phase. There is no fixed stoichiometric ratio of boundary lipids and oligomers. Between 24k and 28k lipid molecules are able to surround each isolated oligomer composed of k Ca2+-ATPase monomers. The equations are also used to analyze DPH studies on DPPC bilayers containing Ca2+-ATPase for T < Tc. The results reported are in accord with the predictions of the model. An analytical expression for probabilities is in very good agreement with the results of computer simulation.

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