Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes.

Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R9 and K9 peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na+ and Ca2+ salt concentrations, showing that the apparent strength of adsorption of R9 decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R9 and K9, we first demonstrated that the binding of R9 to POPC is tighter by almost 2 orders of magnitude compared to that of K9. Finally, upon the addition of an excess of either Na+ or Ca2+ ions with R9, the total fluorescence correlation signal is lost, which implies the unbinding of R9 from the PC bilayer, in agreement with our predictions from MD simulations.

Complex ion effects on polypeptide conformational stability: chloride and sulfate salts of guanidinium and tetrapropylammonium.

The effects of chloride and sulfate salts of tetrapropylammonium (TPA(+)) and guanidinium (Gdm(+)) on the conformational stabilities of tryptophan zipper (trpzip) and α-helical (alahel) peptides were measured by circular dichroism spectroscopy. Like Gdm(+), TPA(+) interacts with the planar tryptophan indole group, perturbing the conformational stability of trpzip peptides. TPA(+) effects are largely unaffected by sulfate, indicating an absence of the heteroion pairing that is observed in concentrated Gdm(2)SO(4) solutions. TPA(+) stabilizes helical conformations in alahel peptides, indicating exclusion from the peptide bond. The observations are broadly consistent with predictions of molecular dynamics simulations [Mason, P. E.; et al. J. Phys. Chem. B2009, 113, 3227-3234], indicating that the effects of complex ions on proteins are increasingly predictable in terms of ion hydration, complementary interactions with specific protein groups, and ion-pairing contributions.

Specificity of ion-protein interactions: complementary and competitive effects of tetrapropylammonium, guanidinium, sulfate, and chloride ions.

The interactions of ions with a model peptide (a single melittin alpha-helix) in solutions of tetrapropylammonium sulfate or guanidinium chloride were examined by molecular dynamics simulations. The tetrapropylammonium cation shares the geometrical property of essentially flat faces with the previously examined guanidinium cation, and it was found that that this geometry leads to a strong preference for tetrapropylammonium to interact in a similar stacking-type fashion with flat nonpolar groups such as the indole side chain of tryptophan. In contrast to guanidinium, however, tetrapropylammonium does not exhibit strong ion pairing or clustering with sulfate counterions in the solution. Sulfate was found to interact almost exclusively and strongly with the cationic groups of the peptide, such that, already in a 0.1 m solution of tetrapropylammonium sulfate, the 6+ charge of the peptide is effectively locally neutralized. In combination with previous simulations, neutron scattering studies, and experiments on the conformational stability of model peptides, the present results suggest that the Hofmeister series can be explained in higher detail by splitting ions according to the effect they have on hydrogen bonding, salt bridges, and hydrophobic interactions in the protein and how these effects are altered by the counterion.