Interaction of Arginine-Rich Cell-Penetrating Peptides with an Artificial Neuronal Membrane

Abstract

Arginine-rich cell-penetrating peptides (RRCPPs) exhibit intrinsic neuroprotective effects on neurons injured by acute ischemic stroke. Conformational properties, interaction, and the ability to penetrate the neural membrane are critical for the neuroprotective effects of RRCCPs. In this study, we applied circular dichroism (CD) spectroscopy and coarse-grained molecular dynamics (CG MD) simulations to investigate the interactions of two RRCPPs, Tat(49-57)-NH₂ (arginine-rich motif of Tat HIV-1 protein) and PTD4 (a less basic Ala-scan analog of the Tat peptide), with an artificial neuronal membrane (ANM). CD spectra showed that in an aqueous environment, such as phosphate-buffered saline, the peptides mostly adopted a random coil (PTD4) or a polyproline type II helical (Tat(49-57)-NH₂) conformation. On the other hand, in the hydrophobic environment of the ANM liposomes, the peptides showed moderate conformational changes, especially around 200 nm, as indicated by CD curves. The changes induced by the liposomes were slightly more significant in the PTD4 peptide. However, the nature of the conformational changes could not be clearly defined. CG MD simulations showed that the peptides are quickly attracted to the neuronal lipid bilayer and bind preferentially to monosialotetrahexosylganglioside (DPG1) molecules. However, the peptides did not penetrate the membrane even at increasing concentrations. This suggests that the energy barrier required to break the strong peptide-lipid electrostatic interactions was not exceeded in the simulated models. The obtained results show a correlation between the potential of mean force parameter and a peptide's cell membrane-penetrating ability and neuroprotective properties.

Keywords: PTD4 peptide; Tat(49–57)-NH2; artificial neuronal membrane; cell-penetrating peptides; circular dichroism studies; liposome; molecular dynamics simulations.

Figure 1

CD spectra showing the influence of Ala-scan substitution (Lys^50,51,Arg^52,55,57→Ala) on the conformational properties of Tat(49–57)-NH₂.

Figure 2

CD spectra showing the conformational behavior of Tat(49–57)-NH₂ (A) and PTD4 (B) peptides during their interaction with ANM liposomes.

Figure 3

Formation of hydrophilic pores in pulling CG MD simulations of (A) Tat(49–57)-NH₂ and (B) PTD4 across the neuronal membrane model. (C) Comparison of potential mean of force (PMF) between Tat(49–57)-NH₂ and PTD4. The headgroups of POPC, POPE, POPS and DPSM are colored gray, lime, yellow and red, respectively. The sugar part of DPG1 is in magenta, while lipid acyl chains are presented in grey. CHOL is presented in blue as CPK model. The amino acid residues in licorice representation are colored as follows: Arg-blue, Lys-cyan, Gln-orange, Tyr-green and Ala-grey.

Figure 4

Representative snapshots from the POPC:POPE:POPS:DPG1:DPSM:CHOL binding CG MD simulations for multipeptide systems with Tat(49–57)-NH₂ (A) and PTD4 (B). The headgroups of POPC, POPE, POPS, and DPSM are colored gray, lime, yellow, and red, respectively. The sugar part of DPG1 is in magenta, while lipid acyl chains are presented in gray. CHOL is presented in blue as CPK model.

Figure 5

(A) 2D density map of DPG1 lipids and PTD4 in the upper leaflet of the membrane. The grid spacing was set to 1 Å. The last 1 µs of 10-µs CG MD simulations was considered for the analysis. (B) Partial density and charge density profiles averaged over the last 1 µs of MD CG simulations of the systems without peptide and with multiple Tat(49–57)-NH₂ and PTD4 molecules.