Characterization of electrostatic and nonelectrostatic components of protein--membrane binding interactions.

A general method was developed to determine the thermodynamic parameters for the interaction of protein with membranes. Protein intrinsic tryptophan fluorescence was quenched by titration with large unilamellar vesicles containing 9,10-dibrominated distearoylphosphatidylcholine (Br4-DSPC) or a small amount of trinitrophenylphosphatidylethanolamine (TNP-PE), Binding was modeled as a bimolecular reaction of free protein with a unit of "n" lipid molecules and a dissociation constant, Kd. The contribution of residual fluorescence and light scattering could be eliminated by using the second derivative of the titration function as the basis for calculations. For the binding of C-terminal channel domain polypeptides(178-190 residues) of the colicin El ion channel, n=50-60 and Kd=2-3 nM at pH 4, ionic strength, I=0.12 M, and anionic lipid content = 40% (surface potential, psi o =-30 mV), conditions for which the protein has high activity. Values of n = 95 and 210 for the binding of a C-terminal 293-residue colicin fragment and the 522 residue intact colicin E1 molecule scale qualitatively according to the increase in molecular size. General methods are presented to distinguish the electrostatic (delta G el) and nonelectrostatic (delta G nel) components of the total delta G for binding. Using Br4DSPC as the quencher, the binding of the channel polypeptide, P178, was characterized by delta G approximately -9.8 kcal/mol, and delta G el approximately -7.0 kcal/mol, and delta G el= -2.8 kcal/mol (psi o = -30 mV). Using TNP-PE as the quencher, similar values of delta G approximately -9.3 to -9.9 kcal/mol were determined, a somewhat smaller value for delta G nel approximately -5.0 kcal/mol, and a correspondingly larger value for deltaGnel approximately -4.9 kcal/mol. The existence of a delta G nel component of this magnitude may distinguish proteins that have the potential to insert into the membrane.