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. 2021 Mar;50(2):127-142.
doi: 10.1007/s00249-021-01503-x. Epub 2021 Mar 4.

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

Sarah F Verbeek  1 , Neha Awasthi  2 , Nikolas K Teiwes  1 , Ingo Mey  1 , Jochen S Hub #  3   4 , Andreas Janshoff #  1
Affiliations

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

Sarah F Verbeek et al. Eur Biophys J. 2021 Mar.

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[Formula: see text] and of an arginine side chain mimic R[Formula: see text] to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R[Formula: see text] but not R[Formula: see text] reduces the line tension of a membrane with anionic lipids. While R[Formula: see text] peptides form a layer on top of a partly negatively charged lipid bilayer, R[Formula: see text] leads to its disintegration. Complementary, our simulations show R[Formula: see text] causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R[Formula: see text] 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[Formula: see text] and R[Formula: see text] but not short R[Formula: see text] reduce the free energy of nucleating a pore. In short R[Formula: see text], the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer [Formula: see text] are membrane-active.

Keywords: Arginine; Breakthrough force; CPP; MD-simulation.

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Figures

Fig. 1
Fig. 1
a Stick representation of an arginine side chain mimic RSide and oligo-arginines R2, R4, R8 and R12. b Simulation snapshot of a POPG membrane with an open pore. Lipid headgroups are represented as yellow spheres, tails as grey sticks. For clarity, only water molecules near the pore are shown as red/white spheres. Arginine-8 is shown as colored green/blue/white/red spheres. c Exemplary force vs. distance curve with a breakthrough event, as collected on the atomic force microscope, with illustrations of the cantilever tip and membrane. (1) As the cantilever approaches, its deflection is initially zero. The contact point (2) is usually defined as the point from which cantilever deflection starts increasing. The cantilever tip deforms the bilayer as it exerts more force (3), until at some point it breaks through with a discrete event with two kinks (4). After breakthrough, the cantilever is in contact with the solid support (5). The force at which the breakthrough event occurs is defined as ’yield force’, in our case the average force of plateau (4)
Fig. 2
Fig. 2
Empirical cumulative distribution function of yield force F as obtained on the three investigated bilayers using the same cantilever. Medians of data distributions are indicated with dotted lines
Fig. 3
Fig. 3
Boxplots of normalized yield forces Fn of membranes varying in composition after incubation with different R-derivatives. Normalization was performed for the median of the yield force obtained in the absence of R-derivatives for each experiment. Each boxplot contains combined data from at least two independent experiments. The bottom and top edge of the boxplots indicate the 25th and 75th percentile of each data set, and the line dividing the box indicates the median. The upper and lower whisker represent approximately 2.7 standard deviations higher or lower than the mean, respectively. Outliers (points beyond 2.7 standard deviations from the mean) are shown as grey points. a POPC; b POPC:POPE 1:1; c POPC:POPG 1:1. d Normalized histograms of yield force results of POPC:POPG 1:1 and a combination with RSide and R12, respectively, all probed with the identical cantilever. The probability density function of each data histogram was fitted (red lines) to a continuum nucleation model as introduced by Loi et al. (Loi et al. 2002), to obtain line tension Γ and spreading pressure S
Fig. 4
Fig. 4
Exemplary leakage assays of sulforhodamine B from pure POPG vesicles. With lower concentrations of RSide or R9 than indicated or with POPC:POPG 1:1, no leakage was achieved (data not shown)
Fig. 5
Fig. 5
Exemplary RIfS experiments illustrating the different behavior of RSide and R-oligopeptides. Change in bulk concentration is indicated by blue dashed lines. a RSide added to a POPC:POPG 1:1 membrane, the membrane was dissolved at the second addition of peptide as the membrane thickness decreases; (b/c) step-wise increase in R8-concentration in the presence of a POPC:POPG 1:1 bilayer (b) and POPC (c). No significant increase in optical thickness and hence no binding is observed in the absence of negative charges on the bilayer
Fig. 6
Fig. 6
PMFs of pore formation across a pure-lipid membranes composed of POPG, POPE, or POPC (see legend) and b POPG membranes containing poly-arginine or arginine side chains
Fig. 7
Fig. 7
Modulation of membrane properties by the addition of polyarginines, derived from MD simulations: a free energy of pore nucleation, b membrane thickness, and c area per lipid. The lipid is encoded by color
Fig. 8
Fig. 8
Pore formation during MD simulations over membranes of (a–c) pure POPG and (d–f) of POPG plus eight R8. The columns in shaded colors show the mass density of (from left to right) headgroups, water, tails, and R8, as averaged over umbrella sampling windows restrained at (A/D) ξch=0.225, (B/E) ξch=0.625, and ξch=1, corresponding states with a flat membrane, a partial defect, and a fully formed pore, respectively. Densities are plotted as function of lateral distance r and vertical distance z from the center of the defect. Evidently, R8 accumulates at the partial and fully formed defect (white arrows), where R8 replaces water and stabilizes lipid headgroups (cyan arrows)
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