TranslocationThermodynamics of Linear and CyclicNonaarginine into Model DPPC Bilayer via Coarse-Grained MolecularDynamics Simulation: Implications of Pore Formation and Nonadditivity

摘要

Structural mechanisms and underlying thermodynamic determinants of efficient internalization of charged cationic peptides (cell-penetrating peptides, CPPs) such as TAT, polyarginine, and their variants, into cells, cellular constructs, and model membrane/lipid bilayers (large and giant unilamellar or multilamelar vesicles) continue to garner significant attention. Two widely held views on the translocation mechanism center on endocytotic and nonendocytotic (diffusive) processes. Espousing the view of a purely diffusive internalization process (supported by recent experimental evidence, [Säälik, P.; et al. J. Controlled Release>2011, 153, 117–125]), we consider the underlying free energetics of the translocation of a nonaarginine peptide (Arg9) into a model DPPC bilayer. In the case of the Arg9 cationic peptide, recent experiments indicate a higher internalization efficiency of the cyclic structure (cyclic Arg9) relative to the linear conformer. Furthermore, recent all-atom resolution molecular dynamics simulations of cyclic Arg9 [Huang, K.; et al. Biophys. J., >2013, 104, 412–420]suggested a critical stabilizing role of water- and lipid-constitutedpores that form within the bilayer as the charged Arg9 translocatesdeep into the bilayer center. Herein, we use umbrella sampling moleculardynamics simulations with coarse-grained Martini lipids, polarizablecoarse-grained water, and peptide to explore the dependence of translocationfree energetics on peptide structure and conformation via calculationof potentials of mean force along preselected reaction paths allowingand preventing membrane deformations that lead to pore formation.Within the context of the coarse-grained force fields we employ, weobserve significant barriers for Arg9 translocation frombulk aqueous solution to bilayer center. Moreover, we do not findfree-energy minima in the headgroup–water interfacial region,as observed in simulations using all-atom force fields. The pore-formingpaths systematically predict lower free-energy barriers (ca. 90 kJ/mollower) than the non pore-forming paths, again consistent with all-atomforce field simulations. The current force field suggests no preferencefor the more compact or covalently cyclic structures upon enteringthe bilayer. Decomposition of the PMF into the system’s componentsindicates that the dominant stabilizing contribution along the pore-formingpath originates from the membrane as both layers of it deformed dueto the formation of pore. Furthermore, our analysis revealed thatalthough there is significant entropic stabilization arising fromthe enhanced configurational entropy exposing more states as the peptidemoves through the bilayer, the enthalpic loss (as predicted by theinteractions of this coarse-grained model) far outweighs any formerstabilization, thus leading to significant barrier to translocation.Finally, we observe reduction in the translocation free-energy barrierfor a second Arg9 entering the bilayer in the presenceof an initial peptide restrained at the center, again, in qualitativeagreement with all-atom force fields.