Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance

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Abstract
The selectivity of interaction between bovine spinal cord myelin basic protein (MBP) and eight different spin-labeled lipid species in complexes with dimyristoylphosphatidylglycerol (DMPG) and between spin-labeled phosphatidylglycerol and spin-labeled phosphatidylcholine in complexes of MBP with various mixtures of DMPG and dimyristoylphosphatidylcholine (DMPC) has been studied by electron spin resonance (ESR) spectroscopy. In DMPC/DMPG mixtures, the protein binding gradually decreased with increasing mole fraction of DMPC in a nonlinear fashion. The lipid-protein binding assays indicated a preferential binding of the protein to phosphatidylglycerol relative to phosphatidylcholine without complete phase separation of the two lipids. The outer hyperfine splittings (2Amax) of both phosphatidylglycerol and phosphatidylcholine labeled at C-5 of the sn-2 chain (5-PGSL and 5-PCSL, respectively) were monitored in the lipid-protein complexes as a function of the mole fraction of DMPC. The increases in the value of Amax induced on binding of the protein were larger for 5-PGSL than for 5-PCSL, up to 0.25 mole fraction of DMPC. Beyond this mole fraction the spectral perturbations induced by the protein were similar for both lipid labels. The ESR spectra of phosphatidylglycerol and phosphatidylcholine labeled at C-12 of the sn-2 chain were two component in nature, indicating a direct interaction of the protein with the lipid chains, at mole fractions of DMPC up to 0.25. Quantitation of the motionally restricted spin-label population by spectral subtraction again indicated a preferential interaction of the protein with phosphatidylglycerol relative to phosphatidylcholine. Up to DMPC mole fractions of 0.25, the microenvironment of the protein was enriched in DMPG. The interaction of MBP with phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), diphosphatidylglycerol (CL), and diacylglycerol (DG), spin-labeled on C-5 of the sn-2 chain, was studied in complexes with DMPG. The protein-induced increases in the outer hyperfine splitting of the spin-label ESR spectra established a sequence for the selectivity of interaction with the MBP in the order PS- > CL- > PA2- > PG- > PI- > PA- > PE.+-. > PC.+-. > DG. Assuming fast exchange for the ESR spectra of the C-5 labels, relative association constants of the different lipids with the MBP were determined. pH titration of the PA spin-label in DMPG complexes revealed a stronger interaction with the protein of the doubly negatively charged than of the singly charged species, and the shift in the pK of the PA spin-label indicated a partial dehydration of the DMPG lipid surface on binding of the protein.