Modulatory Effects of Acidic pH and Membrane Potential on the Adsorption of pH-Sensitive Peptides to Anionic Lipid Membrane

Abstract

Anionic lipid membrane electrostatic potential and solution pH can influence cationic peptide adsorption to these bilayers, especially those containing simultaneously acid and basic residues. Here, we investigate the effects of the pH solution on MP1 (IDWKKLLDAAKQIL-NH2) adsorption to anionic (7POPC:3POPG) lipid vesicles in comparison to its analog H-MP1, with histidines substituting lysines. We used the association of adsorption isotherms and constant pH molecular dynamic simulations (CpHMD) to explore the effects of membrane potential and pH on peptides' adsorption on this lipid membrane. We analyzed the fluorescence and zeta potential adsorption isotherms using the Gouy-Chapman theory. In CpHMD simulations for the peptides in solution and adsorbed on the lipid bilayer, we used the conformations obtained by conventional MD simulations at a μs timescale. Non-equilibrium Monte Carlo simulations provided the protonation states of acidic and basic residues. CpHMD showed average pKa shifts of two to three units, resulting in a higher net charge for the analog than for MP1, strongly modulating the peptide adsorption. The fractions of the protonation of acidic and basic residues and the peptides' net charges obtained from the analysis of the adsorption isotherms were in reasonable agreement with those from CpHMD. MP1 adsorption was almost insensitive to solution pH. H-MP1 was much more sensitive to partitioning, at acidic pH, with an affinity ten times higher than in neutral ones.

Keywords: CpHMD; fluorescence spectroscopy; membrane potential; pH-responsive peptides; zeta potential.

Figure 1

(a) Representative emission spectra of 2 μM MP1 in buffer and the presence of POPC:POPG (7:3) LUVs at the indicated concentration, at pH 5.5 and 25 °C. (b) Binding isotherms of MP1 (black) or H-MP1 (light gray) to 7POPC:3POPG LUVs at pHs 5.5 (circles), 6.5 (squares), and 7.4 (triangles). The solid lines represent the non-linear fit represented in Equation (3). (c) Partition constant (K${}_p$) obtained from (b). All data correspond to the average (±SD) of three independent experiments.

Figure 2

Binding isotherms of MP1 (black) and H-MP1 (gray) to 7POPC:3POPG LUVs at pHs 5.5 (circles), 6.5 (squares), and 7.4 (triangles). The symbols represent the experimental results obtained from the fluorescence assay, and continuous lines represents the best theoretical isotherms obtained by Gouy–Chapman theory.

Figure 3

(a) Zeta potential ($\zeta$) measurements of 7POPC:3POPG LUVs as a function of the peptide-to-lipid molar ratio ([P]/[L]${}_{1/2}$, considering half of the lipid content) for MP1 (dark symbols) and H-MP1 (gray symbols). Circles, squares and triangles represent pHs 5.5, 6.5, and 7.4, respectively. The opened symbols represent the condition with a slight increase in vesicle diameter (see Figure S1). All data correspond to the average (±SD) of two independent experiments. (b) Surface potential ($\psi$) calculated from $\zeta$ values (down triangles), fluorescence (stars), and GC theory (continuous lines).

Figure 4

Arrangement of the titratable residues in MP1 and its analog in solution and adsorbed on the lipid bilayer. The distances show the salt bridges’ network. The backbone is shown as a cartoon in gray. The side chains are shown in black, the acidic in red, and the basic in cyan. Distances are measured in angstroms. The radial distribution function g(r) of the residues pair Lys-Asp for MP1 and His-Asp for H-MP1 are shown in Figure S4.