How arginine derivatives alter the stability of lipid membranes: dissecting the roles of side chains, backbone and termini

Sarah F Verbeek,Ingo Mey,Nikolas K Teiwes,Neha Awasthi,Jochen S Hub,Andreas Janshoff

doi:10.1007/s00249-021-01503-x

Sarah F Verbeek, Ingo Mey + Show 4 more

Open Access

https://doi.org/10.1007/s00249-021-01503-x

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Abstract

Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R_n and of an arginine side chain mimic R_mathrm {Side} to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R_mathrm {Side} but not R_n reduces the line tension of a membrane with anionic lipids. While R_n peptides form a layer on top of a partly negatively charged lipid bilayer, R_mathrm {Side} leads to its disintegration. Complementary, our simulations show R_mathrm {Side} causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R_8 similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R_n and R_mathrm {Side} but not short R_n reduce the free energy of nucleating a pore. In short R_n, the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer mathrm {R}_{n} are membrane-active.

Highlights

Cell-penetrating peptides (CPPs) are able to cross cell membranes, without being cytotoxic
We studied pore formation using two quantitative, complementary methods: (1) atomic force microscopy (AFM) on solid-supported lipid bilayers in phosphate buffer and (2) free-energy calculations of pore formation based on all-atom molecular dynamics (MD) simulations (Fig. 1)
In experiment and in simulation, model membranes were composed of common lipids (POPC, POPE, POPG), which we studied under the influence of arginine oligopeptides of different lengths or of an arginine side chain mimic (N-propylguanidine or R Side ), see Fig. 1a

Summary

Introduction

Cell-penetrating peptides (CPPs) are able to cross cell membranes, without being cytotoxic. Development of novel CPPs for the purpose of drug delivery or for neuroprotective applications have gained enormous interest in recent decades (Gasparini et al 2015; Guidotti et al 2017; Gestin et al 2017; Milletti 2012; Kauffman et al 2015; Raucher and Ryu 2015; Langel 2019; Foerg and Merkle 2008; Koren and Torchilin 2012; Alvaro et al 2016; Ruseska and Zimmer 2020). Polyarginines may act as CPPs and perform better as a CPP than TAT derivatives (Wender et al 2000; Mitchell et al 2000). CPPs vary greatly in sequence, most of them are overall cationic or amphipathic (Milletti 2012).

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