Design of Peptides for Membrane Insertion: The Critical Role of Charge Separation

Abstract

A physical understanding of membrane permeation and translocation by small, positively charged molecules can illuminate cell penetrating peptide mechanisms of entry and inform drug design. We have previously investigated the permeation of the doubly charged peptide WKW and proposed a defect-assisted permeation mechanism where a small molecule with +2 charge can achieve a metastable state spanning the bilayer by forming a membrane defect with charges stabilized by phospholipid phosphate groups. Here, we investigate the membrane permeation of two doubly charged peptides, WWK and WWWK, with charges separated by different lengths. Through complementary experiments and molecular dynamics simulations, we show that membrane permeation was an order of magnitude more favorable when charges were separated by an ∼2–3 Å greater distance on WWWK compared to WWK. These results agree with the previously proposed defect-assisted permeation mechanism, where a greater distance between positive charges would require a less extreme membrane defect to stabilize the membrane-spanning metastable state. We discuss the implications of these results in understanding the membrane permeation of cell-penetrating peptides and other small, positively charged membrane permeants.

The Structures of Heterogeneous Membranes and Their Interactions with an Anticancer Peptide: A Molecular Dynamics Study

“…Membranes are highly viscous fluids with strong interactions between their components and significant dynamic and static correlations. The findings of the formation of spatial and temporal microdomains in membranes demonstrate those correlations [ 10 , 11 , 49 , 51 , 52 ]. To examine correlations beyond pairs ( Figure 5 ) we plotted distributions of three body distances from the phospholipids to the center of mass of the peptide ( Figure 8 and Figure 9 ).…”

“…The charges of the peptide (red spheres) remain close to the membrane surface. As we indicated in our previous studies [ 23 , 48 , 49 , 50 ], significant membrane distortions are required to allow the charges to pass the hydrophobic core and to switch to the other side of the membrane. These events did not occur in the relatively short simulations we conducted here.…”

Leaflet-Dependent Effect of Anionic Lipids on Membrane Insertion by Cationic Cell-Penetrating Peptides

“…On the basis of previous studies, it is likely that all three peptides are, to some extent, translocating from the exterior to the interior of LUVs on the time scale of our experiments. 15,36 If so, the peptides would be able to insert into both leaflets of the bilayer and, thus, should be impacted by the anionic lipid composition of both leaflets. This effect may explain the slight high-energy shift of R 6 W 3 and WWWK in LUVs with 10 mol % outer leaflet anionic lipid and 30 mol % inner leaflet anionic lipid compared to vesicles with 10 mol % anionic lipid in both leaflets, as the inner leaflet composition of the asymmetric LUV would promote insertion of translocated peptides back into the membrane.…”

“…We have recently shown that permeants can traverse membranes through a defect-assisted mechanism in which a cationic peptide can cross the hydrophobic region of the bilayer by increasing membrane hydration and interacting with inner and outer leaflet lipid headgroups and phosphates. − Furthermore, anionic lipids can affect CPP–membrane interactions and insertion in a headgroup-dependent manner. , A recent study from our group using an anticancer CPP, NAF-1 44–67 , showed that phosphatidic acid (PA) lipids enhanced membrane insertion and translocation to a greater extent than phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids . Computational studies also suggest that this peptide permeates deeper in membranes with binary mixtures of PA or PS and zwitterionic phosphatidylcholine (PC) and locally enriches its environment with anionic lipids .…”

“…Aliphatic NAF-1 44–67 has a charged, unstructured C-terminal segment and, when interacting with membranes, a hydrophobic helix at the N-terminal segment. , The tetramer WWWK-NH 2 has a net charge of +2 and does not have any significant secondary structure. We recently proposed defect-assisted permeation mechanisms for both NAF-1 44–67 and WWWK in PC membranes where free energy barriers (20 and 15 kcal mol –1 , respectively) for the peptides traversing the headgroup/phosphate region are bridged by a metastable state with the peptides permeating into the membrane and defect-associated lipids accommodating positive charges in the membrane core along with water molecules. − R 6 W 3 is shorter than NAF-1 44–67 and less structured but has a comparable net charge of +6. This peptide forms a short helix when interacting with membranes where the tryptophan residues are immersed in the hydrophobic region of the membrane on one side of the helix and the arginine residues at the membrane–aqueous interface on the other side of the helix. , R 6 W 3 has a high affinity for anionic lipids, locally clustering PG lipids when interacting with membranes, and is hypothesized to permeate membranes through lipid reorganization and induced curvature. − …”