Measurement of Hanatoxin-Induced Membrane Thinning with

Mar 5, 2017 - Lamellar X-ray diffraction (LXD) was applied on stacked planar bilayers in ... LiouPo-Huang LiangPei-Ming ChenStephen A. HoltIsaac Furay...
0 downloads 0 Views 1MB Size
Article pubs.acs.org/Langmuir

Measurement of Hanatoxin-Induced Membrane Thinning with Lamellar X‑ray Diffraction Meng-Hsuan Hsieh,† Yu-Shuan Shiau,‡ Horng-Huei Liou,§,∥ U-Ser Jeng,⊥ Ming-Tao Lee,*,⊥,# and Kuo-Long Lou*,†,‡ †

Institute of Biotechnology, National Taiwan University, Taipei 10672, Taiwan Membrane Protein Research Core, Center for Biotechnology, National Taiwan University, Taipei 10672, Taiwan § Division of Neurology, National Taiwan University Hospital, Taipei 10002, Taiwan ∥ Institute of Pharmacology, National Taiwan University, Taipei 10051, Taiwan ⊥ National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan # Department of Physics, National Central University, Jhongli 32001, Taiwan ‡

ABSTRACT: Membrane perturbation induced by cysteine-rich peptides is a crucial biological phenomenon but scarcely investigated, in particular with effective biophysical-chemical methodologies. Hanatoxin (HaTx), a 35-residue polypeptide from spider venom, works as an inhibitor of drk1 (Kv2.1) channels, most likely by interacting with the voltage-sensor. However, how this water-soluble peptide modifies the gating remains poorly understood, as the voltage sensor was proposed to be deeply embedded within the bilayer. To see how HaTx interacts with phospholipid bilayers, we observe the toxin-induced perturbation on POPC/DOPG-membranes through measurements of the change in membrane thickness. Lamellar X-ray diffraction (LXD) was applied on stacked planar bilayers in the near-fully hydrated state. The results provide quantitative evidence for the membrane thinning in a concentration-dependent manner, leading to novel and direct combinatory approaches by discovering how to investigate such a biologically relevant interaction between gating-modifier toxins and phospholipid bilayers.



INTRODUCTION It is well recognized that many single-helical peptides can bind to the membrane−water interface and/or intercalate into the nonpolar chain region.1−3 In our previous work on the quantitative parameters obtained from the oriented circular dichroism (OCD) and lamellar X-ray diffraction (LXD) measurements for the single helical peptides, we take the pore-forming activity of peptides one step closer to the description at molecular levels. These parameters can be used to obtain the threshold concentration required for the pore formation2 and will be useful for molecular dynamics simulations. In addition, our analyses of peptide−lipid interactions and on the elucidation of cell membrane compositions are required to solve the riddle of the cell-type selectivity by way of discussions on how to interpret lipiddependence of membrane thinning.2 Compared to the aforementioned knowledge on singlehelical peptides, there is no description for the biophysics in the membrane partitioning of cysteine-rich proteins, such as hanatoxin (HaTx1, or in general, HaTx)4 (Figure 1). Hanatoxin, a 35-residue toxin isolated from Chilean tarantula, has been one of the most extensively studied peptides used to characterize the blocking properties of voltage-gated potassium channel Kv2.1 through surface binding and mutational studies between toxins and channels.5−8 According to the Paddle model,9 the Kv channel voltage sensors are proposed to be © XXXX American Chemical Society

Figure 1. Structural comparison of HaTx in different orientations. Molecular surface of HaTx. Molecules in different orientations are generated by rotations for 90° as shown. Hydrophobic residues are colored in white, whereas basic and acidic residues are colored in blue and in red, respectively.

buried deep inside the membrane hydrocarbon core. How can HaTx reach the Kv2.1 voltage sensor from extracellular to form gating inhibitions? The most critical feature on the HaTx surface is the hydrophobic patch and the surrounding charged residues that form the so-called “charged belt”.4 Such characteristics on the protein surface indeed predict the ability of HaTx to exist in an amphipathic interface by interacting with the membrane phospho groups through charged residues and presumably Received: January 8, 2017 Revised: February 27, 2017 Published: March 5, 2017 A

DOI: 10.1021/acs.langmuir.7b00064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Hippel.16 Examinations on the correct folding of the synthetic HaTx was performed with circular dichroism (CD) spectrometry (J-810, JASCO Inc., Tokyo, Japan). Lamellar X-ray Diffraction (LXD) Data Acquisition and Analysis. Toxins and lipids of various molar ratios were prepared in trifluoroethanol and chloroform 1:1 (v/v). The homogeneous mixtures of lipid and toxins in the form of oriented multilayers, a stack of parallel lipid bilayers on a silicon wafer from the solution of different peptide-to-lipid molar ratio (P/L); the sample films were vacuumed dried and then hydrated via water vapor. The preparation of such oriented samples and the sample chamber for LXD were the same as those used in our previous studies,1,17 except that the relative humidity was controlled via a temperature−humidity chamber. The temperature was set to 30 °C. In addition to the measurement at 98.5% relative humidity (RH), a series of measurements was performed at lower levels of humidity regarding the purpose of phase determination. Precise RH reading for these lower levels of humidity was not necessary because the swelling method18,19 for phase determination may use the lamellar-repeat spacings as the variables. LXD was measured with a synchrotron X-ray diffractometer (13A1, National Synchrotron Radiation Research Center (NSRRC), Taiwan). The equilibrium of the sample at each humidity setting was ensured by an agreement between the averages of at least three subsequently analyzed diffraction patterns. Only samples that produced at least five discernible diffraction peaks were accepted. Each peptide−lipid combination was measured with at least three separately prepared samples. Each sample was measured twice apart for at least 10 h to check the reproducibility. The procedure for data reduction was described in our previous papers.2,3 Briefly, the procedure started with a background removal followed by the absorption and diffraction volume corrections. Subsequently, the integrated peak intensities were corrected for the polarization and the Lorentz factors.18 The magnitude of the diffraction amplitude was the square root of the integrated intensity. The phases were determined by the swelling method.18,19 With their phases determined, the diffraction amplitudes were Fourier-transformed to obtain the trans-bilayer electron density profiles. The profiles were not normalized to the absolute scale; instead they gave the correct phosphate-to-phosphate (ptp) distances, since the latter are independent of the normalization.20

with acyl chains of fatty acids through hydrophobic/aromatic residues. This may also imply the ability for residues of such toxins to find their lipid interaction partners via conformational adjustments during the binding process and via insertions into the membrane environment.4,10−12 Taken together, the gatingmodifier spider toxins like HaTx can alter the gating potentials required for the voltage-gated potassium (Kv) channels to open via the involvements of phospholipids.13,14 However, the physical-chemical investigations on the interaction details between HaTx and phospholipid bilayers are so far entirely absent. Thus, we examine in this study the membrane thinning as an index for perturbations on POPC/DOPG bilayers (Figure 2) induced by HaTx binding with LXD on stacked planar

Figure 2. Structures of lipid molecules. (A) 1-Palmitoyl-2-oleoyl-snglycero-3-phosphocholine, POPC. (B) 1,2-Dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt), DOPG.

bilayers. LXD provides not only an experimental demonstration but also an effective analysis on membrane thickness in high accuracy.





RESULTS To increase the resolution for thickness measurement, LXD studies were conducted. The samples used in this method were in the form of oriented multilayers, a stack of parallel lipid bilayers on a solid substrate. As shown in Figure 3A, the individual diffraction patterns were obtained at a constant value for pressure with the molar ratio of peptide to lipid (P/L) equal to 1/50, all at 30 °C as well as 98.5% relative humidity (RH = 98.5%). Since the diffraction patterns may deteriorate at higher RH values due to undulated fluctuations of the membranes, the phasing problems in obtaining high-resolution electron density profile could be overcome by choosing a Fourier-constructed form factor from the diffraction amplitudes measured at one humidity level which went through all the data points, as shown in Figure 3B. When the phase determination is accomplished, the amplitudes from the diffraction patterns will be used to construct the trans-bilayer electron density profiles (Figure 3C). More results for a series of P/L ratios are also shown in Figure 3D as peak-to-peak distances derived from the electron density profiles for the bilayer. In the case of HaTx and POPC/ DOPG-membranes (Figure 3), the ptp distance decreases by about 0.6 Å corresponding to the change in P/L from 0 to 1/ 100 and decreases by about 1.0 Å with the increase in P/L ratio from 0 to 1/50, as shown in Figure 3D. This clearly demonstrates a saturation of binding.

MATERIALS AND METHODS

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DOPG) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Toxin Production. Wild-type hanatoxin (HaTx) was either purified from Grammostola spatula venom (Spider Pharm Inc. Yarnell, AZ) as described in Swatz and MacKinnon, and Ruta and MacKinnon7,15 or synthesized with an Applied Biosystems model 433A peptide synthesizer. For the latter, the linear precursors were synthesized by solid-phase methodology with Fmoc chemistry, starting from Fmoc-Ser-preloaded resin by a variety of blocking groups for the protection of the amino acids. After trifluoroacetic acid cleavage, a crude linear peptide was extracted with 2 M acetic acid and diluted to a final concentration of 25 μM. A solution containing 0.1 M ammonium acetate, 2 M urea, and 2.5 mM reduced/0.25 mM oxidized glutathione was adjusted to pH 7.8 with aqueous NH4OH and stirred slowly at 4 °C for 3 days. The folding reaction was monitored with reverse-phase high-performance liquid chromatography (RP-HPLC), and the crude oxidized product was purified by successive chromatography steps with CM-cellulose CM-52 and preparative RP-HPLC with a C18 silica column. The purity of the synthetic HaTx was confirmed by analytical RP-HPLC and matrix-assisted laser desorption ionization-time-offlight mass spectrometry (MALDI-TOF-MS) operations. Concentration of HaTx was determined from dry weight of the protein. To confirm the toxin concentrations we also measured absorbance at 280 nm and calculated the concentrations of the toxin with an extinction coefficient of 8.6 × 103 M−1 cm−1 as suggested by Gill and von B

DOI: 10.1021/acs.langmuir.7b00064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

Figure 3. Toxin-induced membrane thinning measured with LXD. (A) Representative X-ray diffraction patterns for POPC/DOPG (2:1) phospholipid bilayer (P/L (peptide-to-lipid molar ratio) = 1/50) at the highest hydration level (98.5% RH) in the presence or absence of HaTx. (B) Phasing diagrams for the X-ray diffraction of POPC/DOPG (2:1) bilayer containing HaTx at P/L = 1/50. Phases are chosen according to the swelling methods.18,19 (C) Fourier-constructed electron density profile for a POPC/DOPG (2:1) bilayer containing HaTx at P/L = 1/50. (D) Peakto-peak (ptp) distance versus P/L for POPC/DOPG (2:1) bilayer in the presence of HaTx. Error bars represent deviations from three independent measurements.



DISCUSSION In our previous work on the quantitative parameters obtained from the OCD and LXD measurements, we have derived the threshold concentration required for the pore formation and solved the riddle of the cell-type selectivity by way of discussions on how to interpret the lipid-dependence of membrane thinning.2,3 Compared with such peptide insertions, however, HaTx was proposed to interact with phospholipid membranes in the absence of pore formation. Therefore, membrane partitioning of cysteine-rich proteins indeed requires to be regarded as a special focus when studying the protein− membrane interactions. To expand the knowledge and to address an unanswered question for channel gating regarding the interactions between HaTx and phospholipids, we performed a series of biophysical observations to provide structural evidence for the membrane partitioning of HaTx. Lamellar X-ray diffraction (LXD)2 was applied to measure the change in thickness of POPC/DOPG membranes in the presence of HaTx. Our LXD results showed that the thickness of POPC/DOPG membranes was modified upon HaTx binding from 37.42 to36.46 Å. Because the surface coverage rates for toxins on membranes have been taken into consideration, quantification of membrane thinning is revealed to be precise and accurate. Therefore, in our study, we applied planar multilayer system to examine the interactions between an invading toxin and phospholipid bilayer. LXD measurements may provide higher signal-to-noise ratio for two reasons: (1) LXD increases the resolution for data through signal amplifications by diffraction. (2) Instead of being scattered by vesicles, LXD provides more

unambiguous boundaries between proteins, lipid molecules and solution. Nevertheless, our results have clearly demonstrated the objective parameters necessary to illustrate the membrane thinning induced by the binding of HaTx, a gating-modifier cysteine-rich peptide. For the LXD measurements, in the humidity region from 98% RH to 100% RH, the D spacing is sensitive to hydration21 but the bilayer thickness is insensitive to hydration.17,22 In this study, the D spacing more than 57 Å indicates the hydration is more than 98% RH and again we are in attempt to emphasize the systematical thickness change in the same hydration level rather than the absolute value under fully hydrated conditions. Consequently, LXD results show that the state in a near-fully hydration is accurate enough to represent the behavior of a bilayer in excess of water environments. When HaTx molecules were only slightly attached on the surface of the membranes, the estimated ∼12 Å molecular diameter might result in an increase in the membrane thickness, instead of an apparent thinning. As a consequence, a related assumption could be also applied on the condition of a lesser partitioning (less deep), in which HaTx was supposed to be located at the membrane interface only for those areas with curvature. LXD results might represent a binding status for an intact and a more effective partitioning into the deeper part of bilayer, such as hydrocarbon core. So that the 0.2−0.4 Å magnitude of thinning difference can be regarded as a consequence of the absence of the aforementioned additional scattering components. This implies conclusively that HaTx partitions into the phospholipid acyl chains, rather than only at membrane interface, which will be verified in the future with C

DOI: 10.1021/acs.langmuir.7b00064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir neutron reflectivity or diffractions by measuring the penetration depth(s) of HaTx inside the phospholipid bilayers. Furthermore, we compared the trans-bilayer electron density profiles (EDP) of POPC/DOPG-membranes without and with HaTx (Figure 4) to figure out structural change of bilayer

Figure 5. Normalized area expansion, ΔA/A, versus P/L. ΔA/A is equal to −Δh/h, as calculated from Figure 3D using the relation h = ptp − 10 Å. The linear fitting indicated by dashed line gives the following equation: y = 2.0559x + 0.0012 (R2 = 0.9863). The slope of fitting line corresponds to (AP/AL).

sectional area of HaTx has been estimated according to NMR structure4 to be approximately 616 Å2. It is interesting to note that the measured AP values (140 Å2) is smaller than the cross sections of the peptides. Such smaller AP values, as compared with the peptide cross sections, can be actually explained, if considering some water molecules released from the headgroup region when the peptide is embedded. The extent of peptide partitioning is classified as (1) binding in the headgroup region on the bilayer surface is termed as surface binding; (2) penetrating into the hydrocarbon region of the bilayer is referred to as dipping.

Figure 4. Structural modifications of bilayer induced by HaTx binding. Trans-bilayer electron density profiles constructed from measured diffraction amplitudes, displayed for pure POPC/DOPG-membranes (black line) and P/L = 1/50 (red line), respectively. Dash arrow indicates the main peak for the phosphate group, and solid arrow indicates the turning point for the double-chain methylenes of the bilayer.

induced by HaTx binding. As shown in Figure 4, EDPs have the conventional feature for a bilayer structure: a main peak for the phosphate group (dash arrow) and turning point (solid arrow) for the double-chain methylenes on each side of the bilayer and a central trough for the methyl terminals. The phosphate-tophosphate (ptp) distance of bilayer with HaTx is smaller than the bilayer without HaTx, i.e., the bilayer thinning induced by HaTx binding. Correspondingly, the EDP of a bilayer with HaTx has steeper slope around the turning point. This feature induced by HaTx binding is similar to hydration process shown in Figure 3C. In the hydration process, the water molecules penetrate into the space in-between phosphate groups to make hydrocarbon chains more disordered23 and consequently the steeper slope around the turning point was shown. Accordingly, HaTx not only binds to bilayer surface but also partitions into the interface to cause hydrocarbon chains more disordered. According to the calculations in the previous and present studies,24 the hydrocarbon thickness, h, of the bilayer was estimated by subtracting twice the length of the glycerol region (from the phosphate to first methylene of the hydrocarbon chain), i.e., 10 Å, from the ptp distance.25,26 The fractional increase in the monolayer area due to peptide binding is ΔA/A = (AP/AL) × (P/L), where P/L is the bound peptide-to-lipid molar ratio, AL is the cross-sectional area of the lipids (AL for POPC is 68 Å2),25 and AP is the area of expansion induced by one peptide. In a bilayer, the acyl-chains belong to an elastic region. Due to volume conservation of this region, we were able calculate the normalized area expansion by the relation, −Δh/h = ΔA/A, where h is the thickness of the hydrophobic region and A is the membrane area. Indeed, we have found that in all cases the bilayer thickness decreases linearly with an increased P/L. Thus, the slope of ΔA/A versus P/L extracted from Figure 3D gives the value of AP (Figure 5). The lengthwise cross-



CONCLUSIONS Previously, our studies have delineated the mechanism of membrane thinning-effects induced by the binding of singlehelical peptides. Critical and quantitative parameters to determine the types of pore formation on membranes were also disclosed. In this study, we apply lamellar X-ray diffraction to describe the molecular mechanism for membrane thinning with systems containing HaTx, a cysteine-rich protein, and POPC/DOPG compositions of phospholipids. HaTx interacts directly with POPC/DOPG-membranes without pore formation or membrane disruptions. Apparently HaTx was predicted to use the charge-belt to interact with phosphogroups of membranes and use the hydrophobic patch to insert itself into the bilayers, and, implied from our results, on acylchains. Our present work has for the first time provided biophysical-chemical analyses for the perturbations of HaTx on the membrane. Further structural-functional investigations based on our results will allow the in-depth observations on the dynamic scenario of HaTx−Kv2.1 interaction details within the membrane phospholipids.



AUTHOR INFORMATION

Corresponding Authors

*(M.-T.L.) Tel: +886 35780281 ext.7109. Fax: +886 35783813. E-mail: [email protected]. *(K.-L.L.) Tel: +886 233665857. E-mail: [email protected]; [email protected]. ORCID

Meng-Hsuan Hsieh: 0000-0003-1414-5975 D

DOI: 10.1021/acs.langmuir.7b00064 Langmuir XXXX, XXX, XXX−XXX

Article

Langmuir

(15) Ruta, V.; MacKinnon, R. Localization of the voltage-sensor toxin receptor on KvAP. Biochemistry 2004, 43, 10071−9. (16) Gill, S. C.; von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182, 319−26. (17) Ludtke, S.; He, K.; Huang, H. Membrane thinning caused by magainin 2. Biochemistry 1995, 34, 16764−9. (18) Blaurock, A. E. Structure of the nerve myelin membrane: proof of the low-resolution profile. J. Mol. Biol. 1971, 56, 35−52. (19) Torbet, J.; Wilkins, M. H. X-ray diffraction studies of lecithin bilayers. J. Theor. Biol. 1976, 62, 447−58. (20) Wu, Y.; He, K.; Ludtke, S. J.; Huang, H. W. X-ray diffraction study of lipid bilayer membranes interacting with amphiphilic helical peptides: diphytanoyl phosphatidylcholine with alamethicin at low concentrations. Biophys. J. 1995, 68, 2361−9. (21) Kucerka, N.; Liu, Y.; Chu, N.; Petrache, H. I.; Tristram-Nagle, S.; Nagle, J. F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles. Biophys. J. 2005, 88, 2626−37. (22) Harroun, T. A.; Heller, W. T.; Weiss, T. M.; Yang, L.; Huang, H. W. Experimental evidence for hydrophobic matching and membranemediated interactions in lipid bilayers containing gramicidin. Biophys. J. 1999, 76, 937−45. (23) Hung, W. C.; Chen, F. Y.; Huang, H. W. Order-disorder transition in bilayers of diphytanoyl phosphatidylcholine. Biochim. Biophys. Acta, Biomembr. 2000, 1467, 198−206. (24) Lee, M. T.; Chen, F. Y.; Huang, H. W. Energetics of pore formation induced by membrane active peptides. Biochemistry 2004, 43, 3590−9. (25) Nagle, J. F.; Tristram-Nagle, S. Structure of lipid bilayers. Biochim. Biophys. Acta, Rev. Biomembr. 2000, 1469, 159−95. (26) Hung, W.-C.; Chen, F.-Y. The hydrophobic-hydrophilic interface of phospholipid membranes studied by lamellar X-ray diffraction. Chin. J. Phys. 2003, 41, 85−91.

U-Ser Jeng: 0000-0002-2247-5061 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We are very thankful for the careful reading of manuscripts by Prof. Betty W. Shen and the crucial advice from Profs. Johann Deisenhofer and Kirsten Fischer-Lindl during the second Formosan Symposium on Structural Biology of Membrane Proteins and Biomembranes in Taipei (2012). We also appreciate the helpful discussions with Profs. Chikashi Toyoshima and Stephen White at the NRPB symposia (2012/2013). This work was supported in part by the Taiwanese NSC funding for grants 102-2320-B-002-034, 992324-B-002-004-MY2, 98-2324-B-002-005, 94-2320-B-002-123, 94-2317- B-002-019, and 92-2311-B-002-101 and NSRRC travel funds (2009/2011-1-P752, 2010-2-P1302, 2012/0-2P1399, and N-2014-1-020) for K.-L.L.



REFERENCES

(1) Chen, F. Y.; Lee, M. T.; Huang, H. W. Evidence for membrane thinning effect as the mechanism for peptide-induced pore formation. Biophys. J. 2003, 84, 3751−8. (2) Huang, H. W.; Chen, F. Y.; Lee, M. T. Molecular mechanism of Peptide-induced pores in membranes. Phys. Rev. Lett. 2004, 92, 198304. (3) Lee, M. T.; Hung, W. C.; Chen, F. Y.; Huang, H. W. Mechanism and kinetics of pore formation in membranes by water-soluble amphipathic peptides. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 5087− 92. (4) Takahashi, H.; Kim, J. I.; Min, H. J.; Sato, K.; Swartz, K. J.; Shimada, I. Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins. J. Mol. Biol. 2000, 297, 771−80. (5) Swartz, K. J.; MacKinnon, R. Hanatoxin modifies the gating of a voltage-dependent K+ channel through multiple binding sites. Neuron 1997, 18, 665−73. (6) Swartz, K. J.; MacKinnon, R. Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K+ channels. Neuron 1997, 18, 675−82. (7) Swartz, K. J.; MacKinnon, R. An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. Neuron 1995, 15, 941−9. (8) Li-Smerin, Y.; Swartz, K. J. Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel. J. Gen. Physiol. 2000, 115, 673−84. (9) Jiang, Y.; Lee, A.; Chen, J.; Ruta, V.; Cadene, M.; Chait, B. T.; MacKinnon, R. X-ray structure of a voltage-dependent K+ channel. Nature 2003, 423, 33−41. (10) Huang, P. T.; Shiau, Y. S.; Lou, K. L. The interaction of spider gating modifier peptides with voltage-gated potassium channels. Toxicon 2007, 49, 285−92. (11) Revell Phillips, L.; Milescu, M.; Li-Smerin, Y.; Mindell, J. A.; Kim, J. I.; Swartz, K. J. Voltage-sensor activation with a tarantula toxin as cargo. Nature 2005, 436, 857−60. (12) Huang, P. T.; Chen, T. Y.; Tseng, L. J.; Lou, K. L.; Liou, H. H.; Lin, T. B.; Spatz, H. C.; Shiau, Y. Y. Structural influence of hanatoxin binding on the carboxyl terminus of S3 segment in voltage-gated K(+)-channel Kv2.1. Recept. Channels 2002, 8, 79−85. (13) Nishizawa, M.; Nishizawa, K. Interaction between K+ channel gate modifier hanatoxin and lipid bilayer membranes analyzed by molecular dynamics simulation. Eur. Biophys. J. 2006, 35, 373−81. (14) Milescu, M.; Vobecky, J.; Roh, S. H.; Kim, S. H.; Jung, H. J.; Kim, J. I.; Swartz, K. J. Tarantula toxins interact with voltage sensors within lipid membranes. J. Gen. Physiol. 2007, 130, 497−511. E

DOI: 10.1021/acs.langmuir.7b00064 Langmuir XXXX, XXX, XXX−XXX