Exchange rates at the lipid-protein interface of myelin proteolipid protein studied by spin-label electron spin resonance

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Abstract
The electron spin resonance (ESR) spectra from spin-labeled phospholipids in recombinants of myelin proteolipid apoprotein with dimyristoylphosphatidylcholine have been simulated with the exchanged-coupled Bloch equations to obtain values for both the fraction of motionally restricted lipids and the exchange rate between the fluid and motionally restricted lipid populations. The rate of exchange between the two spin-labeled lipid components is found to lie in the slow exchange regime of nitroxide ESR spectroscopy. The values obtained for the fraction of motionally restricted component in the exchange-coupled spectra are found to be in good agreement with those obtained previously by spectral subtraction for the same system [Brophy, P.J., Horvath, L. I., and Marsh, D. (1984) Biochemistry 23, 860-865]. The rate of lipid exchange off the protein is independent of lipid/protein ratio for a given spin-labeled phospholipid, as expected, and decreases with increasing selectivity of the various phospholipids for the protein. At 30.degree.C and for ionic strength 0.1 and pH 7.4, the off-rate constants are 4.6 .times. 106 s-1 for phosphatidic acid, 1.1 .times. 107 s-1 for phosphatidylserine, 1.6 .times. 107 s-1 for phosphatidylcholine, and 2.2 .times. 107 s-1 for phosphatidylethanolamine. These values are in the inverse ratio of the relative association constants of the various lipids for the protein (Brophy et al., 1984) and are appreciably slower than the rate of lipid diffusion in dimyristoylphosphatidylcholine bilayers. The exchange rate increases with increasing temperature, with an activation energy of 24 kJ .cntdot. mol-1 for spin-labeled phosphatidic acid in the fluid phase and with a steeper rate of increase in the phase transition region. These results strongly support the ESR spectral subtraction methods used previously to analyze the thermodynamics of lipid-protein interactions.