Membrane interaction of erythroid spectrin: Surface-density-dependent high-affinity binding to phosphatidylethanolamine

Abstract

Density-dependent spectrin binding to dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine (DMPC/DMPE) small uni-lamellar vesicles (SUVs) has been directly evaluated in this work from the increase in the extent of quenching of the tryptophan fluorescence of spectrin at two different temperatures, above and below the main phase transition temperatures (T_m). Results from the binding studies of spectrin to phospholipid SUVs indicated that the binding dissociation constant K_d, increased from 45±7 nM in pure DMPC SUVs to 219±20 nM in DMPC/DMPE (50:50) SUVs, both in the gel and liquid crystalline phase. However, in pure DMPE SUVs the K_d decreased drastically to 0.7±0.2 nM in the gel phase at 18°C and to 2.6±0.7 nM in the fluid phase at 55°C indicating a high affinity binding of spectrin for the bilayer-forming DMPE. The maximum extent of phospholipid-induced quenching and the number of spectrin molecules associated with one SUV particle, evaluated in the present work, led to a model in DMPC/DMPE bilayer membranes indicating the PE-binding site of spectrin to localize at one of the terminal domains of the dimeric spectrin. A direct evidence of the localization of the PE-binding site at one of the terminal ends of the spectrin dimer also came from electron microscopic observation in fluid membranes made of bovine brain PE.

• Erythroid spectrin
• phospholipids binding
• phosphatidylethanolamine
• fluorescence

PL, phospholipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidylethanolamine; SUVs, small uni-lamellar vesicles; K_d, dissociation constant of spectrin binding to phospholipid vesicles; K_d^m, dissociation constant of spectrin binding to phospholipid monomers; K_app, apparent affinity constant of spectrin binding to phospholipid vesicles; K_app^m, apparent affinity constant of spectrin binding to phospholipid monomers; M_PL, molecular mass of phospholipid monomers; N_PL, number of phospholipid monomers associated with one spectrin dimmer; M_SUV, molecular mass of the phospholipid vesicles; Q_max, maximum extent of fluorescence quenching; T_m, phase transition temperature; DPH, diphenylhexatriene; CD, circular dichoirism

PL, phospholipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidylethanolamine; SUVs, small uni-lamellar vesicles; K_d, dissociation constant of spectrin binding to phospholipid vesicles; K_d^m, dissociation constant of spectrin binding to phospholipid monomers; K_app, apparent affinity constant of spectrin binding to phospholipid vesicles; K_app^m, apparent affinity constant of spectrin binding to phospholipid monomers; M_PL, molecular mass of phospholipid monomers; N_PL, number of phospholipid monomers associated with one spectrin dimmer; M_SUV, molecular mass of the phospholipid vesicles; Q_max, maximum extent of fluorescence quenching; T_m, phase transition temperature; DPH, diphenylhexatriene; CD, circular dichoirism