Abstract
There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.