Cell penetrating peptides (CPPs) can cross cell membranes in a receptor independent manner and transport cargo molecules inside cells. These peptides can internalize through two independent routes: energy dependent endocytosis and energy independent translocation across the membrane, but the exact mechanisms are still unknown. The interaction of the CPP with different membrane components is certainly a preliminary key point that triggers internalization, such as the interaction with lipids to lead to the translocation process. In this study, we used two arginine-rich peptides, RW9 (RRWWRRWRR-NH2), which is a potent CPP, and RL9 (RRLLRRLRR-NH2) that, although binding tightly and accumulating on membranes, does not enter into cells. Using a set of experimental and theoretical techniques, we studied the binding, insertion and orientation of the peptides into different model membranes as well as the subsequent membrane reorganization. Herein we show that although the two peptides had rather similar behavior regarding lipid membrane interaction, subtle differences were found concerning the depth of peptide insertion, effect on the lipid chain ordering and kinetics of peptide insertion in the membrane, which altogether might explain their different cell internalization capacities. Molecular dynamics simulation studies show that some peptide molecules flipped their orientation over the course of the simulation such that the hydrophobic residues penetrated deeper in the lipid core region while Arg-residues maintained H-bonds with the lipid headgroups, serving as a molecular hinge in a conformation that appeared to correspond to the equilibrium one.

Cell penetrating peptides (CPPs) are able to cross membranes without using receptors but only little information about the underlying mechanism is available. In this work, we investigate the interaction of the two arginine-rich CPPs RW9 and RL9 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and POPC/POPG membranes with varying POPG content using isothermal titration calorimetry (ITC), solid-state nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics (MD) simulations. Both peptides were derived from the known CPP penetratin and it was shown previously that RW9 is able to penetrate membranes better than RL9. Overall, the results show that both RW9 and RL9 have a relatively small influence on the membrane. They increase the order of the lipids in the headgroup region and reduce order in the acyl chains indicating that they are located in the lipid/water interface. In addition, the flexibility of the membrane is slightly increased by both peptides but RW9 has a larger influence than RL9. The differences observed in the influences on POPC and POPG as well as MD simulations on the mixed POPC/POPG bilayers of 850ns length each show that both peptides preferentially associate with and enrich the charged PG lipids almost 2fold in an area of 12Å around the peptides. As expected, we could not observe any membrane crossing on the simulation time scale of 850ns but observed that some peptides flipped their orientation during binding to the membrane. Interestingly, all observed flips coincided with structural changes in the peptides indicating that structural changes or flexibility might play a role during the binding of arginine-rich CPPs to membranes.

We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations—as well as their extensive fluctuations—suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin.

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