Free Energy Analyses of Cell-Penetrating Peptides Using the Weighted Ensemble Method

Abstract

Cell-penetrating peptides (CPPs) have been widely used for drug-delivery agents; however, it has not been fully understood how they translocate across cell membranes. The Weighted Ensemble (WE) method, one of the most powerful and flexible path sampling techniques, can be helpful to reveal translocation paths and free energy barriers along those paths. Within the WE approach we show how Arg9 (nona-arginine) and Tat interact with a DOPC/DOPG(4:1) model membrane, and we present free energy (or potential mean of forces, PMFs) profiles of penetration, although a translocation across the membrane has not been observed in the current simulations. Two different compositions of lipid molecules were also tried and compared. Our approach can be applied to any CPPs interacting with various model membranes, and it will provide useful information regarding the transport mechanisms of CPPs.

Translocation of a single Arg9 peptide across a DOPC/DOPG(4:1) model membrane using the weighted ensemble method

“…This scheme could be applied to the current work to make rapid translocation of Arg 9 or any other CPPs to study their transport mechanisms. Our WE simulations showed that translocation of a single Arg 9 was energetically unfavorable due to much higher potential energy barriers (∼ 1,000 kT) between Arg 9 and the model membrane along the translocation path compared with the previous calculation (∼ 100 kT) from the upper solution to the center of the membrane Choe (2021).…”

“…We found the WE method very effective for studying interactions between Arg 9 and the model membrane and getting free energy barriers along the penetration path. However, we couldn't find a spontaneous translocation of Arg 9 across the membrane because Arg 9 was stuck in the hydrophobic core and couldn't move for a long time Choe (2021).…”

“…Temperature and pressure were kept constant using Langevin dynamics. An external electric field(0.05 V/nm) was applied in the negative z-direction(from CPPs to the membrane) as suggested in previous work Herce et al (2009); Walrant et al (2012) and also in our earlier simulations Choe (2020Choe ( , 2021 to account for the transmembrane potential Roux (2008). The particle-mesh Ewald(PME) algorithm was used to compute the electric forces, and the SHAKE algorithm allowed a 2 fs time step during the simulation.…”

“…When Arg 9 was located at the bottom of the membrane, the WE5 simulation was stopped, and an additional equilibrium simulation (without using the WE method) was run at this position. The equilibrium simulation method is the same as in the Methods section and in the previous work Choe (2020Choe ( , 2021. A total of 1 µs equilibrium simulation was performed.…”

“…Strong interaction with negatively charged phospholipid heads results in inserting R-rich peptides into the lipid bilayer. In the previous study, we implemented a weighted ensemble (WE) method Huber and Kim (1996); Zuckerman and Chong (2017) in all-atom MD simulations of Arg 9 (R9) with a DOPC/DOPG(4:1) model membrane Choe (2021). The WE method is a very flexible path sampling technique, and it is easy to implement in any MD package.…”