Adsorption/Desorption of Cationic-Hydrophobic Peptides on Zwitterionic Lipid Bilayer Is Associated with the Possibility of Proton Transfer

Abstract

Cell-penetrating peptides (CPPs) are short peptides built up from dominantly cationic and hydrophobic amino acid residues with a distinguished ability to pass through the cell membrane. Due to the possibility of linking and delivering the appropriate cargo at the desired location, CPPs are considered an economic and less invasive alternative to antibiotics. Besides knowing that their membrane passage mechanism is a complex function of CPP chemical composition, the ionic strength of the solution, and the membrane composition, all other details on how they penetrate cell membranes are rather vague. The aim of this study is to elucidate the ad(de)sorption of arginine-/lysine- and phenylalanine-rich peptides on a lipid membrane composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipids. DSC and temperature-dependent UV-Vis measurements confirmed the impact of the adsorbed peptides on thermotropic properties of DPPC, but in an inconclusive way. On the other hand, FTIR spectra acquired at 30 °C and 50 °C (when DPPC lipids are found in the gel and fluid phase, respectively) unambiguously confirmed the proton transfer between particular titratable functional groups of R5F2/K5F2 that highly depend on their immediate surroundings (DPPC or a phosphate buffer). Molecular dynamic simulations showed that both peptides may adsorb onto the bilayer, but K5F2 desorbs more easily and favors the solvent, while R5F2 remains attached. The results obtained in this work highlight the importance of proton transfer in the design of CPPs with their desired cargo, as its charge and composition dictates the possibility of entering the cell.

Keywords: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); FTIR and UV-Vis spectroscopy; MD simulations; adsorption mechanism; cell-penetrating peptides R5F2 and K5F2; large unilamellar liposomes (LUV).

Figure 1

Structural formulas of: (a) DPPC (with DPPG headgroup); (b) R5F2; (c) K5F2. Color and gray zig-zag lines denote functional groups that may participate in proton-exchange (see below).

Figure 2

Temperature-dependent UV-Vis spectra (solid curves) and spectral profiles (dotted curves) of: (a) DPPC′ + R5F2; (b) DPPC′ + K5F2; (c) DPPC′. The spectra acquired at 30 °C/50 °C are highlighted (red/wine for DPPC′ + R5F2, blue/navy for DPPC′ + K5F2, gray/dark gray for DPPC′), as well as spectral profile curves (red for DPPC′ + R5F2, blue for DPPC′ + K5F2, dark gray for DPPC′); DSC curves and concentrational profiles of the first principal component accompanied with a double Boltzmann sigmoidal transition of: (d) DPPC′ + R5F2 (wine curve for DSC and red/orange curve for spectral projection of UV-Vis data/double Boltzmann fit); (e) DPPC′ + K5F2 (navy curve for DSC and blue/cyan curve for spectral projection of UV-Vis data/double Boltzmann fit); (f) DPPC′ (dark yellow curve for DSC and dark gray/gray curve for spectral projection of UV-Vis data/double Boltzmann fit). Phase transition temperatures are highlighted with dashed (DSC) and dotted (UV-Vis) lines and are additionally written on graphs and designated with a corresponding color.

Figure 3

Normalized (and smoothed) FTIR spectra of DPPC′ ± R5F2/K5F2 in the following spectral ranges: (a,b) 3000–2820 cm⁻¹; (c,d) 1770–1700 cm⁻¹; (e,f) 1515–1360 cm⁻¹; (g,h) 1130–1020 cm⁻¹; (i,j) 990–940 cm⁻¹. DPPC′ spectra are presented with solid gray/dark gray (30 °C/50 °C) curves, DPPC′ + R5F2 with solid red/wine (30 °C/50 °C) curves, and DPPC′ + K5F2 with solid blue/navy (30 °C/50 °C) curves. The spectra of R5F2/K5F2 are labeled with the same color as corresponding spectra of DPPC′ + R5F2/K5F2 but with dotted curves. Along with the band assignment, their displacement in DPPC′ ± R5F2/K5F2 are designated with light gray-shaded rectangles or solid light gray lines, whereas in R5F2/K5F2 spectra they are marked with yellow rectangles.

Figure 4

The time dependence of the minimum distance of any peptide atom from the phosphorus atom of DPPC for each investigated system in molecular dynamics simulations: (a) K5R2 at 30 °C, (b) R5F2 at 30 °C, (c) K5R2 at 50 °C and (d) R5F2 at 50 °C.

Figure 5

The illustration of possible interactions between the carboxyl group of peptides and DPPC (top) or cationic residues within peptide side chains (bottom). Hydrogen donors are marked in red, and hydrogen acceptors in blue.