Spontaneous Membrane Translocating Peptides: The Role of Leucine-Arginine Consensus Motifs  Taylor Fuselier, William C. Wimley  Biophysical Journal  Volume 113, Issue 4, Pages 835-846 (August 2017) DOI: 10.1016/j.bpj.2017.06.070 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Spontaneous membrane translocation. (A) Given here are example translocation measurements at P/L = 1:100. Lipid vesicles, at 0.5 mM, with entrapped chymotrypsin and external α1-antitrypsin, were incubated with 5-μM NBD-labeled peptide. At intervals, samples were eluted over C18 reverse phase HPLC. The areas of the cleavage product and uncleaved peptides were used to determine the fractional cleavage, as shown. All curves were fit to a single exponential decay. (B) Shown here are the mean and SD of translocation rates in units of h−1. Values are from at least three independent experiments. In all figures and tables, red denotes TP2, and green denotes the LRLLR motif peptide. To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Effect of P/L on activity. (A) Translocation rates were measured as described in Fig. 1 at P/L = 1:100 and P/L = 1:500. For TP2 we also measured the translocation rate for P/L = 1:1000. Peptides for which no bar is shown have rates <0.001 h−1. (B) Shown here is membrane permeabilization across a wide range of P/L for the translocating peptides and motifs. POPC vesicles contained entrapped ANTS and DPX: a fluorophore quencher pair (27). The fluorescence of ANTS was used to quantitate permeabilization. No release of ANTS/DPX was observed for any of the translocating peptides. The pore-forming peptide melittin is a positive control and releases ANTS/DPX at relatively low concentrations. To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Lack of correlation between mole fraction partition coefficient (Kx) and translocation rate (KT). Translocation rate is plotted against mole fraction membrane partition coefficient (Kx), which was measured by fluorescence titration (54) and equilibrium dialysis (28). Linear regression gave a p value of 0.8 for correlation. To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 The effect of transmembrane potential on translocation. POPC vesicles were made with entrapped chymotrypsin and entrapped 128 mM KCl. In the exterior solution, there was α1-antitrypsin and 128 mM NaCl. Valinomycin was added to some samples to shuttle potassium across the membrane and generate a negative inside transmembrane potential of −110 mV. Translocation experiments, as described above, were performed in the absence and presence of 4 nM valinomycin, to create 0 and −110 mV membrane potential, respectively. To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Effect of membrane disorder on translocation. (A) Shown here is the effect of the pore-forming peptide alamethicin on the cleavage of ONEG by chymotrypsin vesicles. Cleavage was measured at 30 min. Below P/L 1:2000, cleavage does not occur, which indicates that peptides cannot enter vesicles through alamethicin pores at P/L ≤ 1:2000. (B) Given here is the translocation rate of peptides at P/L = 1:100 in the absence and presence of alamethicin at P/L = 1:6000, measured as described above. (C) Given here is the translocation rate for TP2 at different concentrations in the presence and absence of alamethicin at P/L = 1:6000. To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Peptide-induced flip-flop. Asymmetric POPC vesicles with 1 mol % C6-NBD PC lipids in the outer monolayer were incubated with peptide at a P/L of 1:25 overnight. A higher P/L was used here to compensate for the weaker binding of the NBD-free peptides compared to translocation experiments done with labeled peptides. A concentration of 45 mM of fresh sodium dithionite was used to quench the accessible NBD on the outer membrane of the vesicles after incubation with buffer, SMTP, or ONEG. Treatment of symmetric vesicles shows that approximately half of the NBD is protected as expected. Each column is the mean ± SD of at least four experiments. By ANOVA, TP2 and LRLLRWC cause highly significant flip-flip (p < 0.0001 and p < 0.001, respectively). The other motif peptides are statistically indistinguishable from blank (p > 0.05). To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 Translocation of concatenated motifs. The fastest (RLRLL) and slowest (LRLRL) motifs were concatenated to form new 12-residue peptides. Both peptides have an RLLR motif at the junction between the motifs. The novel peptides translocated slower than TP2. Translocation rates are not an additive or predictable property in concatenated peptides. To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 8 Effect of arginine side-chain length on translocation. TP2 (Table 1) and variants with shorter and longer arginine side chains were assayed in parallel for translocation as described above. All side chains have a guanidine group, but differ in the number of methylene (-CH2-) groups in the side chain. To see this figure in color, go online. Biophysical Journal 2017 113, 835-846DOI: (10.1016/j.bpj.2017.06.070) Copyright © 2017 Biophysical Society Terms and Conditions