New and Notable
And Yet It Moves Paulo F. Almeida1,* 1
Department of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, North Carolina
E pur si muove (and yet it moves) is a comment attributed to Galileo after the Catholic Church forced him to recant his theory that the earth moved around the sun. In this issue of the Biophysical Journal, Fuselier and Wimley (1) report the discovery of an amino acid sequence motif that appears to impart to polypeptides the ability to translocate across lipid membranes silently—that is, without causing flux of aqueous solutes across the membrane. This work follows a series of articles from Wimley’s laboratory on methods to reliably measure translocation of membraneactive peptides across phospholipid membranes, and on the use of an orthogonal high-throughput assay to screen combinatorial libraries of membrane-active peptides to specifically select for those peptides that spontaneously translocate but do not permeabilize membranes to water-soluble molecules (2–4). The translocating peptide TP2 emerged from this selection procedure as one of the most effective (2). TP2 is a 13-residue cationic peptide with the sequence PLIYLRLLRGQWC-amide. It was shown to cross the membranes of lipid vesicles and cells, even with polar fluorescent dyes attached (2,5). Wimley and co-workers noticed that the central
Submitted June 19, 2017, and accepted for publication July 17, 2017. *Correspondence: [email protected] Editor: Andreas Engel.
sequence in TP2, the motif LRLLR, was present in many of the translocating peptides selected by their screen. They hypothesized that the LRLLR motif could be a minimal sequence that encodes the ability to translocate. Here, Fuselier and Wimley (1) report the test of this hypothesis. They incorporated the motif in the peptide LRLLRWC(NBD)-amide, which is fairly hydrophobic, but bears a þ3 charge in a neutral solution. In addition, a nitrobenzoxadiazole (NBD) fluorophore was attached to the thiol group of the C-terminal Cys (which is amidated). This tag allows monitoring of the cleavage of the Trp-Cys bond by chymotrypsin encapsulated in large unilamellar vesicles of 1-palmitoyl2-oleoylphosphatidylcholine (POPC). Release of Cys-NBD served as the assay for peptide translocation. Further, Fuselier and Wimley tested the effect of the position of the two Arg residues within the LRLLR motif on peptide translocation. TP2 was used as positive control and an observed negative (ONEG), the peptide PLGRPQLRRGQWC-amide was used as negative control for translocation. The authors found that the LRLLR motif translocates better than the parent TP2, but some of its arginine positional variants are even more efficient at crossing the lipid bilayer. Because these peptides cause little membrane perturbation, as evident from the lack of vesicle leakage, it is safe to conclude that pores are not involved in translocation. Furthermore,
experiments were performed at peptide-to-lipid ratios (P/L) as low as 1:500 for the motif peptides and 1:1000 for TP2, suggesting that monomers are the translocating species. Translocation efficiency drops for the motif peptides when P/L is decreased from 1:100 to 1:500, which is typical of membrane-active peptides; but translocation increases for TP2 between P/L of 1:100 and 1:1000, which is unusual. Intuitively, we tend to expect that cationic amphipathic peptides cannot move across a lipid bilayer spontaneously. And yet they move. This is particularly surprising if they cross the bilayer as monomers, without formation of a transient pore that would allow the charges to remain hydrated as the peptide crosses the membrane. We have struggled with this question in our own work on antimicrobial and cell-penetrating peptides (6). The kinetic evidence at low P/L points to membrane translocation by monomers or small oligomers (dimers, trimers). The peptides we have studied are longer and more cationic (20 residues and charges of þ4 to þ7) than those of Fuselier and Wimley, which makes translocation of monomers appear even less plausible (7,8). And although our peptides cause leakage, some also translocate silently, at least initially, across simple POPC bilayers (7). To explain monomer translocation, we have proposed that the rate-limiting step, corresponding to formation of the
http://dx.doi.org/10.1016/j.bpj.2017.07.006 Ó 2017 Biophysical Society.
Biophysical Journal 113, 759–761, August 22, 2017 759
Almeida
transition state (DGz), is peptide insertion from the bilayer surface (interface) to its interior. This is the highest-energy location for the positive charges. Next, however, the peptide can adopt a transmembrane position, in which the charges are stabilized by salt bridges to the lipid phosphate groups (8) or by interaction with water that penetrates the headgroup region of the bilayer. This inserted transmembrane state would have a significantly lower Gibbs energy (DGTM, measured relative to the interface). Once trans-membrane, the peptide is as likely to cross the bilayer as to return to the original side. This idea suggested that placement of the positive charges close to the peptide termini would enhance translocation, a hypothesis that found experimental support (8). Fuselier and Wimley (1) propose a similar idea to stabilize membrane-translocating peptides. Based on Hammond’s postulate, we have estimated DGz by forming the difference DGz z DGoct DGbind, where DGbind is the Gibbs energy of binding from water to the membrane interface and DGoct is the Gibbs energy of transfer from water to octanol (used as a mimic of the bilayer interior), measured by the Wimley-White octanol scale (6,9). None of these quantities include the stabilization of charges by salt bridges or water penetration in the bilayer. Consequently, the values of DGz are quite large. For example, the peptide Ac-TP10W (21 residues, þ4 charge) has DGz z 17 kcal/mol (DGbind ¼ 9.5 and DGoct ¼ þ7 kcal/mol); and Ac-DL1a (26 residues, þ7) has DGz z 22 kcal/mol (DGbind ¼ 8 and DGoct ¼ þ14 kcal/mol) (7,8). The Gibbs energy of the stabilized transmembrane state, however, should be much lower. Indeed, very recent MD simulations by Ulmschneider (10) reveal a simple mechanism that explains much of the experimental observations. In Ulmschneider’s simulations, the 21-residue, cationic (þ5), antimicrobial peptide PGLa translocates spontaneously as monomers across the lipid bilayer without forming pores. The peptides
can remain inserted for microseconds, in a transmembrane orientation stabilized by salt bridges to the phosphates of the lipid headgroups. Both the insertion step and the subsequent completion of translocation are assisted by one or two other nearby peptides, which interact with the translocating monomer, and by water molecules that penetrate the bilayer and help stabilize the cationic amino acid side chains. This assistance by one or two other peptides probably explains the weak concentration dependence observed in the kinetics (which we interpreted as indicative of the involvement of dimers or trimers). It probably explains also the concentration dependence observed by Fuselier and Wimley (1) in the motif peptides, and by many other groups in antimicrobial peptides. Ulmschneider (10) estimated an Arrhenius activation energy of 16 kcal/mol, or DGz z 19 kcal/mol (10), depending on how it is calculated, which compares with our estimates of DGz for Ac-TP10W and Ac-DL1a. Furthermore, in these MD simulations the stabilized inserted state, which lives for microseconds, has a Gibbs energy of DGTM z þ4 kcal/mol relative to the membrane interface. Thus, indeed DGTM DGz. Fuselier and Wimley measured equilibrium binding of TP2, ONEG, and the motif peptides to POPC membranes. Experimentally, for TP2 and the motif peptides, DGbind z 7 kcal/mol (1)— in good agreement with the prediction from the Wimley-White interfacial scale (DGif) for transfer from water to the POPC membrane interface (9). For the LRLLR motif peptides, DGoct ¼ 0 and for TP2, DGoct ¼ 1 kcal/mol. For the negative control ONEG, DGif z 3 and DGoct ¼ þ7 kcal/mol. Thus, we would estimate DGz z 7 kcal/mol for the motif peptides, 6 kcal/mol for TP2, and 10 kcal/mol for ONEG. Note that the motif peptides and TP2 bind well—but not too strongly—to POPC and have a DGz much smaller than the peptides we and Ulmschneider studied. The negative control ONEG has a higher translo-
760 Biophysical Journal 113, 759–761, August 22, 2017
cation barrier than TP2 and the motif peptides and binds very poorly to POPC, explaining why it does not translocate. The rate constant for translocation can be estimated from k ¼ ðD= z a2 ÞeDG =RT , based on Kramers’ theory (11), where D is the diffusion coefficient and a is the characteristic distance of the barrier to be crossed. Using a value D 107 cm2 s1 for diffusion in POPC (12) and a ˚ , the preexponential factor is 10 A 7 10 s1. Therefore, for TP10W (DGz z 17 kcal/mol), we obtain k 105 s1 at room temperature, which corresponds to a characteristic time t 1 day. Experimentally, TP10W translocation occurs in tens of minutes (7), which is 100 times faster. But keep in mind that an error of a factor of 100 in our estimate of D or an error of only 2–3 kcal/mol in our rough estimate of DGz is sufficient to account for the disagreement. For TP2 and the motif peptides, we obtain k 100 s1, or t ¼ 0.01 s. Translocation of TP2 and the motif peptides actually occurs slower than predicted, in minutes to hours (1). Thus, the problem here is to explain the observed slowness of bilayer crossing. Be that as it may, the article by Fuselier and Wimley suggests a recipe to make spontaneously translocating peptides. They must bind well—but not too well—to the membrane (DGbind z 7 kcal/mol) and their inserted state must not have a high energy (DGoct z 0). Thus, the membrane perturbation will probably be small, avoiding leakage, and the translocation energy barrier will be low (DGz z þ7 kcal/mol). This prediction and the simplicity of the motif discovered here open the possibilities for the design of new peptides with the ability to translocate across membranes, as vehicles for the delivery of cargo into cells. ACKNOWLEDGMENTS The author thanks Dr. Jakob Ulmschneider for a preprint of his article.
New and Notable
REFERENCES 1. Fuselier, T., and W. C. Wimley. 2017. Spontaneous membrane translocating peptides: the role of leucine-arginine consensus motifs. Biophys. J. 113:835–846. 2. Marks, J. R., J. Placone, ., W. C. Wimley. 2011. Spontaneous membrane-translocating peptides by orthogonal high-throughput screening. J. Am. Chem. Soc. 133:8995–9004. 3. He, J., K. Hristova, and W. C. Wimley. 2012. A highly charged voltage-sensor helix spontaneously translocates across membranes. Angew. Chem. Int. Ed. Engl. 51:7150–7153. 4. Cruz, J., M. Mihailescu, ., K. Hristova. 2013. Spontaneous membrane-translocating peptides by orthogonal high-throughput screening. Biophys. J. 104:2419–2428.
5. He, J., W. B. Kauffman, ., W. C. Wimley. 2013. Direct cytosolic delivery of polar cargo to cells by spontaneous membranetranslocating peptides. J. Biol. Chem. 288: 29974–29986. 6. Almeida, P. F., and A. Pokorny. 2012. Interactions of antimicrobial peptides with lipid bilayers. In Comprehensive Biophysics, Vol. 5. Edward H. Egelman, editor.. Academic Press, Cambridge, MA, pp. 189–222. 7. Wheaten, S. A., F. D. O. Ablan, ., P. F. Almeida. 2013. Translocation of cationic amphipathic peptides across the membranes of pure phospholipid giant vesicles. J. Am. Chem. Soc. 135:16517–16525. 8. Ablan, F. D., B. L. Spaller, ., P. F. Almeida. 2016. Charge distribution fine-tunes the translocation of a-helical amphipathic pep-
tides across membranes. Biophys. J. 111: 1738–1749. 9. White, S. H., and W. C. Wimley. 1999. Membrane protein folding and stability: physical principles. Annu. Rev. Biophys. Biomol. Struct. 28:319–365. 10. Ulmschneider, J. P. 2017. Charged antimicrobial peptides can translocate across membranes without forming channel-like pores. Biophys. J. 113:73–81. 11. Kramers, H. A. 1940. Brownian motion in a field of force and the diffusion model of chemical reactions. Physica. 7:284–304. 12. Vaz, W. L. C., R. M. Clegg, and D. Hallmann. 1985. Translational diffusion of lipids in liquid crystalline phase phosphatidylcholine multibilayers. A comparison of experiment with theory. Biochemistry. 24: 781–786.
Biophysical Journal 113, 759–761, August 22, 2017 761
And Yet It Moves Paulo F. Almeida1,* 1
Department of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, North Carolina
E pur si muove (and yet it moves) is a comment attributed to Galileo after the Catholic Church forced him to recant his theory that the earth moved around the sun. In this issue of the Biophysical Journal, Fuselier and Wimley (1) report the discovery of an amino acid sequence motif that appears to impart to polypeptides the ability to translocate across lipid membranes silently—that is, without causing flux of aqueous solutes across the membrane. This work follows a series of articles from Wimley’s laboratory on methods to reliably measure translocation of membraneactive peptides across phospholipid membranes, and on the use of an orthogonal high-throughput assay to screen combinatorial libraries of membrane-active peptides to specifically select for those peptides that spontaneously translocate but do not permeabilize membranes to water-soluble molecules (2–4). The translocating peptide TP2 emerged from this selection procedure as one of the most effective (2). TP2 is a 13-residue cationic peptide with the sequence PLIYLRLLRGQWC-amide. It was shown to cross the membranes of lipid vesicles and cells, even with polar fluorescent dyes attached (2,5). Wimley and co-workers noticed that the central
Submitted June 19, 2017, and accepted for publication July 17, 2017. *Correspondence: [email protected] Editor: Andreas Engel.
sequence in TP2, the motif LRLLR, was present in many of the translocating peptides selected by their screen. They hypothesized that the LRLLR motif could be a minimal sequence that encodes the ability to translocate. Here, Fuselier and Wimley (1) report the test of this hypothesis. They incorporated the motif in the peptide LRLLRWC(NBD)-amide, which is fairly hydrophobic, but bears a þ3 charge in a neutral solution. In addition, a nitrobenzoxadiazole (NBD) fluorophore was attached to the thiol group of the C-terminal Cys (which is amidated). This tag allows monitoring of the cleavage of the Trp-Cys bond by chymotrypsin encapsulated in large unilamellar vesicles of 1-palmitoyl2-oleoylphosphatidylcholine (POPC). Release of Cys-NBD served as the assay for peptide translocation. Further, Fuselier and Wimley tested the effect of the position of the two Arg residues within the LRLLR motif on peptide translocation. TP2 was used as positive control and an observed negative (ONEG), the peptide PLGRPQLRRGQWC-amide was used as negative control for translocation. The authors found that the LRLLR motif translocates better than the parent TP2, but some of its arginine positional variants are even more efficient at crossing the lipid bilayer. Because these peptides cause little membrane perturbation, as evident from the lack of vesicle leakage, it is safe to conclude that pores are not involved in translocation. Furthermore,
experiments were performed at peptide-to-lipid ratios (P/L) as low as 1:500 for the motif peptides and 1:1000 for TP2, suggesting that monomers are the translocating species. Translocation efficiency drops for the motif peptides when P/L is decreased from 1:100 to 1:500, which is typical of membrane-active peptides; but translocation increases for TP2 between P/L of 1:100 and 1:1000, which is unusual. Intuitively, we tend to expect that cationic amphipathic peptides cannot move across a lipid bilayer spontaneously. And yet they move. This is particularly surprising if they cross the bilayer as monomers, without formation of a transient pore that would allow the charges to remain hydrated as the peptide crosses the membrane. We have struggled with this question in our own work on antimicrobial and cell-penetrating peptides (6). The kinetic evidence at low P/L points to membrane translocation by monomers or small oligomers (dimers, trimers). The peptides we have studied are longer and more cationic (20 residues and charges of þ4 to þ7) than those of Fuselier and Wimley, which makes translocation of monomers appear even less plausible (7,8). And although our peptides cause leakage, some also translocate silently, at least initially, across simple POPC bilayers (7). To explain monomer translocation, we have proposed that the rate-limiting step, corresponding to formation of the
http://dx.doi.org/10.1016/j.bpj.2017.07.006 Ó 2017 Biophysical Society.
Biophysical Journal 113, 759–761, August 22, 2017 759
Almeida
transition state (DGz), is peptide insertion from the bilayer surface (interface) to its interior. This is the highest-energy location for the positive charges. Next, however, the peptide can adopt a transmembrane position, in which the charges are stabilized by salt bridges to the lipid phosphate groups (8) or by interaction with water that penetrates the headgroup region of the bilayer. This inserted transmembrane state would have a significantly lower Gibbs energy (DGTM, measured relative to the interface). Once trans-membrane, the peptide is as likely to cross the bilayer as to return to the original side. This idea suggested that placement of the positive charges close to the peptide termini would enhance translocation, a hypothesis that found experimental support (8). Fuselier and Wimley (1) propose a similar idea to stabilize membrane-translocating peptides. Based on Hammond’s postulate, we have estimated DGz by forming the difference DGz z DGoct DGbind, where DGbind is the Gibbs energy of binding from water to the membrane interface and DGoct is the Gibbs energy of transfer from water to octanol (used as a mimic of the bilayer interior), measured by the Wimley-White octanol scale (6,9). None of these quantities include the stabilization of charges by salt bridges or water penetration in the bilayer. Consequently, the values of DGz are quite large. For example, the peptide Ac-TP10W (21 residues, þ4 charge) has DGz z 17 kcal/mol (DGbind ¼ 9.5 and DGoct ¼ þ7 kcal/mol); and Ac-DL1a (26 residues, þ7) has DGz z 22 kcal/mol (DGbind ¼ 8 and DGoct ¼ þ14 kcal/mol) (7,8). The Gibbs energy of the stabilized transmembrane state, however, should be much lower. Indeed, very recent MD simulations by Ulmschneider (10) reveal a simple mechanism that explains much of the experimental observations. In Ulmschneider’s simulations, the 21-residue, cationic (þ5), antimicrobial peptide PGLa translocates spontaneously as monomers across the lipid bilayer without forming pores. The peptides
can remain inserted for microseconds, in a transmembrane orientation stabilized by salt bridges to the phosphates of the lipid headgroups. Both the insertion step and the subsequent completion of translocation are assisted by one or two other nearby peptides, which interact with the translocating monomer, and by water molecules that penetrate the bilayer and help stabilize the cationic amino acid side chains. This assistance by one or two other peptides probably explains the weak concentration dependence observed in the kinetics (which we interpreted as indicative of the involvement of dimers or trimers). It probably explains also the concentration dependence observed by Fuselier and Wimley (1) in the motif peptides, and by many other groups in antimicrobial peptides. Ulmschneider (10) estimated an Arrhenius activation energy of 16 kcal/mol, or DGz z 19 kcal/mol (10), depending on how it is calculated, which compares with our estimates of DGz for Ac-TP10W and Ac-DL1a. Furthermore, in these MD simulations the stabilized inserted state, which lives for microseconds, has a Gibbs energy of DGTM z þ4 kcal/mol relative to the membrane interface. Thus, indeed DGTM DGz. Fuselier and Wimley measured equilibrium binding of TP2, ONEG, and the motif peptides to POPC membranes. Experimentally, for TP2 and the motif peptides, DGbind z 7 kcal/mol (1)— in good agreement with the prediction from the Wimley-White interfacial scale (DGif) for transfer from water to the POPC membrane interface (9). For the LRLLR motif peptides, DGoct ¼ 0 and for TP2, DGoct ¼ 1 kcal/mol. For the negative control ONEG, DGif z 3 and DGoct ¼ þ7 kcal/mol. Thus, we would estimate DGz z 7 kcal/mol for the motif peptides, 6 kcal/mol for TP2, and 10 kcal/mol for ONEG. Note that the motif peptides and TP2 bind well—but not too strongly—to POPC and have a DGz much smaller than the peptides we and Ulmschneider studied. The negative control ONEG has a higher translo-
760 Biophysical Journal 113, 759–761, August 22, 2017
cation barrier than TP2 and the motif peptides and binds very poorly to POPC, explaining why it does not translocate. The rate constant for translocation can be estimated from k ¼ ðD= z a2 ÞeDG =RT , based on Kramers’ theory (11), where D is the diffusion coefficient and a is the characteristic distance of the barrier to be crossed. Using a value D 107 cm2 s1 for diffusion in POPC (12) and a ˚ , the preexponential factor is 10 A 7 10 s1. Therefore, for TP10W (DGz z 17 kcal/mol), we obtain k 105 s1 at room temperature, which corresponds to a characteristic time t 1 day. Experimentally, TP10W translocation occurs in tens of minutes (7), which is 100 times faster. But keep in mind that an error of a factor of 100 in our estimate of D or an error of only 2–3 kcal/mol in our rough estimate of DGz is sufficient to account for the disagreement. For TP2 and the motif peptides, we obtain k 100 s1, or t ¼ 0.01 s. Translocation of TP2 and the motif peptides actually occurs slower than predicted, in minutes to hours (1). Thus, the problem here is to explain the observed slowness of bilayer crossing. Be that as it may, the article by Fuselier and Wimley suggests a recipe to make spontaneously translocating peptides. They must bind well—but not too well—to the membrane (DGbind z 7 kcal/mol) and their inserted state must not have a high energy (DGoct z 0). Thus, the membrane perturbation will probably be small, avoiding leakage, and the translocation energy barrier will be low (DGz z þ7 kcal/mol). This prediction and the simplicity of the motif discovered here open the possibilities for the design of new peptides with the ability to translocate across membranes, as vehicles for the delivery of cargo into cells. ACKNOWLEDGMENTS The author thanks Dr. Jakob Ulmschneider for a preprint of his article.
New and Notable
REFERENCES 1. Fuselier, T., and W. C. Wimley. 2017. Spontaneous membrane translocating peptides: the role of leucine-arginine consensus motifs. Biophys. J. 113:835–846. 2. Marks, J. R., J. Placone, ., W. C. Wimley. 2011. Spontaneous membrane-translocating peptides by orthogonal high-throughput screening. J. Am. Chem. Soc. 133:8995–9004. 3. He, J., K. Hristova, and W. C. Wimley. 2012. A highly charged voltage-sensor helix spontaneously translocates across membranes. Angew. Chem. Int. Ed. Engl. 51:7150–7153. 4. Cruz, J., M. Mihailescu, ., K. Hristova. 2013. Spontaneous membrane-translocating peptides by orthogonal high-throughput screening. Biophys. J. 104:2419–2428.
5. He, J., W. B. Kauffman, ., W. C. Wimley. 2013. Direct cytosolic delivery of polar cargo to cells by spontaneous membranetranslocating peptides. J. Biol. Chem. 288: 29974–29986. 6. Almeida, P. F., and A. Pokorny. 2012. Interactions of antimicrobial peptides with lipid bilayers. In Comprehensive Biophysics, Vol. 5. Edward H. Egelman, editor.. Academic Press, Cambridge, MA, pp. 189–222. 7. Wheaten, S. A., F. D. O. Ablan, ., P. F. Almeida. 2013. Translocation of cationic amphipathic peptides across the membranes of pure phospholipid giant vesicles. J. Am. Chem. Soc. 135:16517–16525. 8. Ablan, F. D., B. L. Spaller, ., P. F. Almeida. 2016. Charge distribution fine-tunes the translocation of a-helical amphipathic pep-
tides across membranes. Biophys. J. 111: 1738–1749. 9. White, S. H., and W. C. Wimley. 1999. Membrane protein folding and stability: physical principles. Annu. Rev. Biophys. Biomol. Struct. 28:319–365. 10. Ulmschneider, J. P. 2017. Charged antimicrobial peptides can translocate across membranes without forming channel-like pores. Biophys. J. 113:73–81. 11. Kramers, H. A. 1940. Brownian motion in a field of force and the diffusion model of chemical reactions. Physica. 7:284–304. 12. Vaz, W. L. C., R. M. Clegg, and D. Hallmann. 1985. Translational diffusion of lipids in liquid crystalline phase phosphatidylcholine multibilayers. A comparison of experiment with theory. Biochemistry. 24: 781–786.
Biophysical Journal 113, 759–761, August 22, 2017 761
