Dynamic Structure and Orientation of Melittin Bound to Acidic Lipid

Article pubs.acs.org/JPCB

Dynamic Structure and Orientation of Melittin Bound to Acidic Lipid Bilayers, As Revealed by Solid-State NMR and Molecular Dynamics Simulation Kazushi Norisada,† Namsrai Javkhlantugs,†,‡ Daisuke Mishima,† Izuru Kawamura,† Hazime Saitô,§ Kazuyoshi Ueda,*,† and Akira Naito*,† †

Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan School of Engineering and Applied Sciences, National University of Mongolia, Ulaanbaatar 14201, Mongolia § Department of Life Science, University of Hyogo, Harima Science Garden City, Kamigori, Hyogo 678-1297, Japan ‡

S Supporting Information *

ABSTRACT: Melittin is a venom peptide that disrupts lipid bilayers at temperatures below the liquid-crystalline to gel phase transition temperature (Tc). Notably, the ability of melittin to disrupt acidic dimyristoylphosphatidylglycerol (DMPG) bilayers was weaker than its ability to disrupt neutral dimyristoylphosphatidylcholine bilayers. The structure and orientation of melittin bound to DMPG bilayers were revealed by analyzing the 13C chemical shift anisotropy of [1-13C]-labeled melittin obtained from solid-state 13C NMR spectra. 13C chemical shift anisotropy showed oscillatory shifts with the index number of residues. Analysis of the chemical shift oscillation properties indicated that melittin bound to a DMPG membrane adopts a bent α-helical structure with tilt angles for the N- and Cterminal helices of −32 and +30°, respectively. The transmembrane melittin in DMPG bilayers indicates that the peptide protrudes toward the C-terminal direction from the core region of the lipid bilayer to show a pseudotransmembrane bent α-helix. Molecular dynamics simulation was performed to characterize the structure and interaction of melittin with lipid molecules in DMPG bilayers. The simulation results indicate that basic amino acid residues in melittin interact strongly with lipid head groups to generate a pseudo-transmembrane alignment. The N-terminus is located within the lipid core region and disturbs the lower surface of the lipid bilayer.

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bend.16,17 The kink angle between two helical rods of melittin in methanol, I2−T11 and L13−Q26, is larger than the kink angles found in the crystals.18 Circular-dichroism spectroscopy studies have shown that melittin adopts an α-helical structure when it binds to sodium dodecylsulfate micelles,19,20 and when it binds to dodecylphosphocholine micelles, the α-helical axis is parallel to the micelle/water interface.21 Divergent conformations were shown for the R22−Q26 region of melittin bound to lipid bilayers, as revealed by transferred nuclear Overhauser enhancement.22 At low peptide-to-lipid ratios, the helical segments are oriented parallel to the bilayer planes, as revealed by polarized attenuated total internal reflection-Fourier transform infrared spectroscopy23 and accessibility measurements of spin-labeled melittin.24 Indeed, melittin lies on the membrane surface as studied by 13C NMR in the presence of an aqueous shift reagent25 and by the hydrogen−deuterium exchange rate.26 An X-ray absolute scale refinement study of melittin at a 1 mol % concentration in lipid deposited on curved substrates revealed

INTRODUCTION Melittin, consisting of 26 amino acid residues, GIGAVLKVLTTGLPALISWIKRKRQQ-NH2, is a major component of the venom of the honey bee, Apis mellifera.1 Melittin disrupts natural and artificial lipid membranes2,3 and promotes phospholipid hydrolysis catalyzed by phospholipase A2.4,5 Furthermore, melittin induces fusion of the phospholipid vesicles,6,7 especially at a temperature above the gel to liquidcrystalline-phase transition temperature (Tc), and disruption toward discoidal membrane fragments below Tc.8,9 Melittin also exhibits a voltage-dependent ion channel activity across the lipid bilayers10,11 and is occasionally regarded as a pore-forming peptide by its oligomerization. In aqueous medium, monomeric melittin assumes a randomcoil conformation, whereas it adopts a primarily α-helical structure with increasing NaCl concentration by forming tetramers,12 depending on the concentration of melittin, the ionic strength, and the pH.13,14 In the crystalline state, melittin adopts a similar α-helical structure, which bends in the T11−G12 region with a kink angle of ∼120° in two crystal polymorphs with space groups of P6122 and C2221.15 The peptides form a tetramer in which the hydrophobic side chains face primarily toward the inside of the bend of the helix, and the hydrophilic side chains primarily extend outside the © 2017 American Chemical Society

Received: November 8, 2016 Revised: January 26, 2017 Published: February 6, 2017 1802

DOI: 10.1021/acs.jpcb.6b11207 J. Phys. Chem. B 2017, 121, 1802−1811

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The Journal of Physical Chemistry B

DMPG was purchased from Sigma (St. Louise, MO) and used without further purification. The powder (50 mg) consisting of melittin and DMPG at a peptide-to-lipid molar ratio of 1:10 was dissolved in 2 mL of chloroform and 1 mL of methanol. The solvent was subsequently evaporated in vacuo to prepare a homogeneous film, followed by the removal of the residual solvent under high vacuum. After swelling by the addition of 500 μL of acidic acid buffer (20 mM acidic acid, 100 mM NaCl, pH 5.0), the film was subjected to 20 freeze/thaw cycles. The hydrated melittin−DMPG membrane dispersion samples were then placed in 5 mm (outer diameter) zirconia tubes and hermetically sealed. NMR Measurements. The 31P- and 13C-direct detection with dipolar decoupling (DD) and cross-polarization and magic angle spinning (CP MAS) NMR measurements were performed on a Chemagnetics CMX Infinity-400 NMR spectrometer (Chemagnetics, Fort Collins, CO) operated at 31 P and 13C resonance frequencies of 161.15 and 100.11 MHz, respectively. Free induction decay (FID) signals were obtained after 90° excitation pulses of 5.0 and 5.5 μs duration under the presence of high-power proton decoupling pulses of 50 and 45 kHz amplitudes and repetition times of 2 and 5 s for 31P and 13 C NMR experiments, respectively. In the 31P NMR measurements, the temperature was decreased from 40 to 0 °C in 10 °C decrements and increased from 0 to 40 °C in 10 °C increments. In the 13C NMR analyses, Lorentzian line broadenings of 60, 100, and 30 Hz were applied to the FID signals to obtain 13C NMR spectra at 40 and −60 °C under static conditions and at 40 °C under magic angle spinning conditions before Fourier transformation, respectively. To determine the 13C chemical shift tensors, 13C NMR spectra of the frozen powder samples were measured at −60 °C using CP with a contact time of 1 ms. The principal values of the 13C chemical shift tensors of the carbonyl carbons were determined by analyzing the asymmetric powder patterns. 31P and 13C chemical shift values were externally referred to 0 ppm for the phosphorus of 85% H3PO4 and 176.03 ppm for the carbonyl carbon of glycine from that of tetramethylsilane (TMS), respectively. MD Simulation Procedure. MD simulations were performed using the CHARMM34 program42 to investigate the structure and orientation of melittin inserted in DMPG membranes. Melittin is a cationic amphipathic peptide consisting of 26 amino acid residues. The sequence H3N+GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2 was used in the MD calculations. Melittin has a net charge of +6, and hydrophobic characteristics were shown in the sequence of the N-terminus at pH 5.0 used in the experiments. In contrast, hydrophilic and basic characteristics were observed in the Cterminus from K21 to the end of the peptide. The right-handed α-helical conformation with five positively charged residues of three K and two R was constructed using the same procedure described in our previous reports.35,36,39 The initial melittin structure was oriented perpendicularly to the DMPG membrane surface with the peptide center of mass placed at the center of mass of the bilayer, where 96 DMPG phospholipid molecules arranged in a square 48 × 2 bilayer. Both surfaces of the bilayer were covered with 3743 water molecules, together with ninety NaCl molecules added as counterions to neutralize the entire system. The final model included 1 melittin, 96 lipids, 3743 water molecules, and 90 NaCls with a total of 22 411 atoms in a 5.6 × 5.6 × 7.0 nm3 orthorhombic box with periodic boundary conditions. The

that the helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups.27 At a peptide-to-lipid ratio of >4 mol % in the lipid bilayers, melittin reorients into a transmembrane alignment.27 In a mechanically oriented ditetradecylphosphatidylcholine (DTPC) membrane, melittin was found to form a transmembrane α-helical structure.28 Melittin (9 mol %) in 86% hydrated lipid bilayer vesicles consisting of dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), or dipalmitoylphosphatidylcholine (DPPC) was shown to adopt a pseudotransmembrane α-helical structure.29,30 Furthermore, membrane-bound melittin was shown to rotate rapidly about the axis parallel to the bilayer normal as a result of lateral diffusion. The interhelical angle of the transmembrane helix of melittin in hydrated vesicles was determined to be ∼120° for DLPC and DPPC bilayers. This angle increases to 140° in the hydrated gel state of the DTPC multilayers.31 An attenuated total-reflection infrared analysis also showed that the α-helix of melittin is oriented parallel and perpendicular to the bilayer surface in a hydrated single planar layer and in dry phospholipid multilayers, respectively.32 The structure and orientation of various membrane-bound antimicrobial peptides have been determined using 13C solidstate NMR spectroscopy. The anisotropies of 13C chemical shift values of [1-13C]-labeled residues in helix-forming peptides bound to the lipid bilayers exhibit an oscillatory behavior. This behavior, called “chemical shift oscillation”, depends on the index number of amino acid residues in cases in which the helical axis rotates rapidly around the bilayer normal.29,30 By analyzing chemical shift oscillation data, our group has accurately determined the tilt angles of α-helical melittin,29,30 dynorphin,33 bombolitin II,34 lactoferrampin,35 and alamethicin.36 In addition, molecular dynamics (MD) simulation can provide detailed insights into the structure, orientation, and interactions between peptides and lipids as demonstrated for melittin37,38 and other membrane-bound peptides.34−36,39 Acidic bilayers, in addition to the neutral bilayer, are also important constituents in biomembranes, especially in bacterial membranes. In such cases, it is important to gain insight into the effect of a possible electrostatic interaction between a positively charged site in peptides as in melittin and a negatively charged site in the head group, besides the usual interaction within the hydrophobic environment of the bilayer. We, therefore, aimed to investigate the structure and orientation of melittin bound to acidic dimyristoylphosphatidylglycerol (DMPG) bilayers, to clarify the effect of such interactions in biological membranes.

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MATERIALS AND METHODS Sample Preparation. 9-Fluorenylmethoxycarbonyl (Fmoc) [1-13C]-L-amino acids were synthesized using n-(9fluorenylmethoxycarbonyl) succinimide (Watanabe Chemical Industry, Hiroshima, Japan) and [1-13C]-labeled amino acids (Cambridge Isotope Laboratories, Andover, MA), following the method reported by Paquet.40 Selectively, 13C-labeled melittin at the carbonyl carbon moiety of G3, A4, V5, G12, L16, I17, and I20 was synthesized using an Applied Biosystems (Foster City, CA) 431 A peptide synthesizer with Fmoc solid-phase chemistry using amide resin. The crude peptide that was yielded by a cleavage reaction of the peptide-resin using Reagent K41 was purified by reverse-phase high-performance liquid chromatography. The purity of synthetic melittin was estimated at >95% on the basis of chromatogram analysis. 1803


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anisotropy (Δδ = δ∥ − δ⊥); (δ⊥, Δδ) changed from (−8.52, 25.4) to (−8.16, 24.3) ppm, as summarized in Table S1. When the temperature was lowered further to 20 °C, the chemical shift values (δ⊥, Δδ) changed from (−8.16, 24.3) to (−7.98, 23.9) ppm. These results indicate that the mobility of the melittin−DMPG lipid bilayer increased as a result of the lipid interacting with melittin when the temperature was lowered, as has been observed with melittin−DMPC and melittin−DPPC bilayers.51 Identical decreases of mobility in the melittin− DMPG bilayers were observed in the course of increasing temperature. When the temperature was lowered further to 10 °C, the chemical shift values (δ⊥, Δδ) changed from (−7.98, 23.7) to (−7.81, 23.1) ppm, and an isotropic peak appeared at −0.10 ppm. This result indicates that the lipid bilayers are partially disrupted to form small vesicles undergoing isotropic motion. When the temperature was lowered to 0 °C, a broad isotropic peak at −0.05 ppm was more intense and overlapped with the broad anisotropic peak, indicating the presence of gelphase components (Figure 1). When the temperature was increased from 0 to 20 °C, the isotropic peak disappeared, and the axially symmetric powder pattern was observed. The complete membrane disruption occurred at 10 °C with the melittin−DMPC bilayer system,29,51 whereas partial disruption occurred in the present melittin−DMPG bilayer system. Morphologic changes in the melittin-binding DMPG bilayers were observed using optical microscopy, as shown in Figure S1. At 10 °C, DMPG bilayer vesicles exhibited partial disruption, and the vesicle surfaces appeared rough, whereas the lipid vesicles were not completely disrupted in contrast to the case of melittin−DMPC systems.29,51 When the temperature was lowered to 3 °C, vesicles with rough surfaces did not resolve by forming small disks. In the case of melittin−DMPC systems, the vesicles were completely disrupted and disappeared at 10 °C.29,51 13 C DD-MAS NMR Spectra of Melittin−DMPG Bilayer Systems. Figure 2 shows 13C DD-MAS NMR spectra of melittin molecules singly labeled with [1-13C] at G3, A4, V5, G12, L16, I17, or I20. The melittin molecules were bound to the DMPG bilayers at 40 °C, and the isotropic chemical shift values (δiso) were determined to be 173.5, 174.4, 175.2, 171.6, 176.1, 174.9, and 175.1 ppm, respectively (Table 1). Typical 13 C chemical shift values (δiso) of (α-helix and β-sheet) are

CHARMM all-atom force field was used in the calculations,43,44 and the modified TIP3 force field45 was used for water. The system was initially energy-minimized by constraining all of the main chain torsion angles of melittin to prevent distortion of the helical structure, followed by energy minimization without any constraints. After the minimization, a 30 ns MD simulation was performed on the isobaric− isothermal ensemble with a time step of 1 fs. Nonbonded interactions were calculated using group-based cutoffs with switching functions updated every five time steps. The switching functions were turned on at 1.2 nm and turned off at 1.35 nm. All hydrogen atom bonds were constrained using the SHAKE BONH algorithm.46 Electrostatic interactions were calculated using the Ewald summation method.47 The dielectric constant was set at 1.0. The temperature was set at 313 K, well above the gel to liquid-crystalline phase transition temperature (Tc) for melittin−DMPG, which was controlled using a Nosé− Hoover thermostat. 48,49 Data were visualized using VMD1.9.1.50

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RESULTS AND DISCUSSION Morphologic Changes in Melittin−DMPG Bilayer Systems, As Revealed by 31P NMR Spectroscopy. Figure 1 shows 31P NMR spectra of the melittin−DMPG bilayer systems hydrated with acetic acid buffer recorded at various temperatures. At 40 °C, a 31P NMR spectrum of an axially symmetric powder pattern characteristic of the liquid-crystalline phase was observed. When the temperature was lowered to 30 °C, the chemical shift value of the upper field edge (δ⊥) and the

Figure 1. Effect of variation in temperature on 31P NMR spectra of melittin−DMPG bilayer systems in the hydrated state. Axially symmetric powder patterns were observed at 40, 30, and 20 °C, as characterized by δ∥ and δ⊥ values. Typically, 200 transients were accumulated for each spectrum.

Figure 2. 13C DD-MAS NMR spectra of hydrated melittin−DMPG bilayer systems at 40 °C. A variety of carbonyl carbons were labeled with 13C nuclei. Typically, 10 000 transients were accumulated for each spectrum. Lipid signals are indicated by asterisks (*). 1804


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The Journal of Physical Chemistry B Table 1. 13C Chemical Shift Values of [1-13C]-Labeled Melittin Bound to DMPG Bilayer 13

3

[1- C]Gly [1-13C]Ala4 [1-13C]Val5 [1-13C]Gly12 [1-13C]Leu16 [1-13C]Ile17 [1-13C]Ile20

δiso (ppm)a

structurec

δ⊥ (ppm)a

Δδ (ppm)a

δ11 (ppm)b

δ22 (ppm)b

δ33 (ppm)b

Δδ(γ) (ppm)

173.5 177.7 175.2 171.6 176.1 174.9 175.1

α-helix α-helix α-helix α-helix α-helix α-helix α-helix

177.6 168.2 175.7 173.5 180.5 162.8 168.9

−12.3 28.5 −1.5 −5.7 −13.2 36.3 18.6

244.0 243.1 245.7

184.9 198.1 190.3

91.7 92.0 89.6

−29.4 −2.0 −24.2

242.5 246.7 243.6

191.6 192.5 192.7

94.3 83.4 89.2

−36.4 8.9 −7.7

a δiso, δ⊥, and Δδ values were measured at 40 °C. bδ11, δ22, and δ33 values were measured at −60 °C. cStructures around each amino acid residue are determined by comparing the experimentally obtained δiso values with typical 13C chemical shift values (δiso) of (α-helix and β-sheet), which are reported as (171.6, 168.5), (176.4, 171.8), (174.9, 171.8), (175.7, 170.2), and (174.9, 172.7) for [1-13C]G, A, V, L, and I, respectively.53

reported as (171.6, 168.5), (176.4, 171.8), (174.9, 171.8), (175.7, 170.2), and (174.9, 172.7) for [1-13C]G, A, V, L, and I, respectively.52,53 By comparing the conformation-dependent 13 C chemical shift values52,53 with the experimentally obtained δiso values, it is clearly indicated that melittin bound to DMPG bilayers adopts an α-helical structure over the entire molecule including both the N- and C-terminal helices. Figure 3 shows 13C static DD-MAS spectra of melittin bound to DMPG bilayers in the liquid crystalline phase at 40 °C. An

Figure 4. 13C CP static NMR spectra of hydrated melittin−DMPG bilayer systems at −60 °C. δ11 and δ22 values were obtained from the spectral patterns, and δ33 values were determined from δ11, δ22, and δiso values using the relationship δiso = (δ11 + δ22 + δ33)/3. Typically, 10 000 transients were accumulated for each spectrum.

pattern in the liquid-crystalline phase at 40 °C, asymmetric powder patterns with large anisotropies were observed, and three principal values (δ11, δ22, and δ33) for the [1-13C]-labeled amino acid residues were determined from the spectral patterns in the rigid molecular state, as summarized in Table 1. The δ33 values were obtained from the δ11, δ22, and δiso values using the relationship δiso = (δ11 + δ22 + δ33)/3. Dynamic Structure and Orientation of Melittin Bound to DMPG bilayers, As Revealed by 13C Chemical Shift Anisotropy (CSA) Data. The dynamic structure of α-helical melittin in the DMPG bilayer can be characterized by knowing that the α-helix rotates about the bilayer normal rather than the α-helical axis as shown in Figure S2. This dynamic manner has been justified by observing the axially symmetric powder pattern of the 13C NMR spectra of the carbonyl carbon in the melittin−DMPC, melittin−DLPC, and melittin−DPPC vesicle systems.29,30 Because anisotropies of axially symmetric chemical shifts are significantly changed among different residues, the αhelix does not rotate about the helical axis but about the bilayer normal.29,30 This dynamic manner of melittin in the lipid bilayers is the key factor to analyze chemical shift anisotropy of the individual carbonyl carbons to reveal the dynamic structure and orientation of melittin in lipid bilayers. Rotational motion of the α-helical axis about the bilayer normal induced by lateral diffusion averages the 13C chemical shift tensors of the backbone carbonyl carbons, which changes a large asymmetric powder pattern with an anisotropy of ∼150 ppm in the immobile state (Table 1) to an axially symmetric

Figure 3. 13C DD static NMR spectra of hydrated melittin−DMPG bilayer systems at 40 °C. A variety of carbonyl carbons were labeled with 13C nuclei. Axially symmetric powder patterns were characterized by δ∥ and δ⊥ values. Lipid signals are indicated by asterisks (*). Typically, 10 000 transients were accumulated for each spectrum.

axially symmetric powder pattern with characteristic δ∥ and δ⊥ values was observed. These axially symmetric powder patterns indicate that the α-helix of the melittin molecules undergoes rapid rotation about the DMPG bilayer normal rather than the α-helical axis, as demonstrated previously by observing a significant increase in the δ⊥ peaks of the carbonyl carbon for magnetically oriented melittin−DMPC, melittin−DLPC, and melittin−DPPC bilayer systems.29,30 By analyzing these spectra, 13 C chemical shift anisotropies (Δδ = δ∥ − δ⊥) for individually [1-13C]-labeled amino acid residues were determined, and the results are summarized in Table 1. Notably, the Δδ values differed significantly, indicating that the melittin helical axis largely tilted toward the bilayer normal, as also determined in our previous studies.29,30 Figure 4 shows 13C static CP-MAS spectra of [1-13C]-labeled melittin molecules bound to DMPG bilayers in the immobile state at −60 °C. In contrast to the axially symmetric powder 1805


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Figure 5. Contour plot of RMSD values of experimental and theoretical 13C chemical shift anisotropies. RMSD values increase from blue to red. Yellow dotted lines indicate the minimum RMSD values.

powder pattern with an anisotropy of ∼40 ppm. As described in our previous paper30 and Supporting Information, the 13C CSA of a carbonyl carbon for the ith residue of an α-helical peptide rotating rapidly about the bilayer normal can be expressed by (Δδcal)i =

γi + 1 − γi = −100°

Equation 3 shows that the difference in γ between contiguous residues is 100° because the peptide adopts an α-helical structure. Figure 5 shows a contour plot of the RMSDs against γ(G3) and ζ generated by varying the angles γ (0−90°) and ζ (0−180°) every 1° using Δδ, δ11, δ22, and δ33 values for G3, A4, and V5 in the N-terminal α-helix and the RMADs against γ(L16) and ζ for L16, I17, and I20 in the C-terminal α-helix, as summarized in Table 1. The lowest RMSDs were obtained at γG3 = 71 ± 8° and ζ = 32 ± 4° for the N-terminal α-helix and γL16 = 80 ± 6° and ζ = 30 ± 3° for the C-terminal α-helix as shown by the dotted yellow lines. Fittings were further justified by plotting chemical shift oscillation curves with experimentally obtained data sets, (γi, Δδi(γi)), as shown in Figure 6A, where γ values were evaluated as γ(A4) = γ(G3) − 100°, γ(V5) = γ(G3) − 200°, γ(L17) = γ(L16) − 100°, and γ(I20) = γ(L16) − 400°. Topology and Transmembrane Alignment of Melittin Bound to DMPG Bilayers. On the basis of the symmetry relationship expressed in eq 1, ζ or −ζ cannot be distinguished because of the (3/2) sin2 ζ relation and γ or γ − 180° cannot be distinguished because of the δ11 cos2 γ + δ33 sin2 γ relation. Therefore, the possible (γ, ζ) combinations to show the minimum RMSD value can be determined according to the following relationship:

3 i i sin 2 ζ(δ11i cos2 γi + δ22 sin 2 γi − δ22 ) 2 ⎛ δ i + δ33i ⎞ i ⎟ + ⎜δ22 − 11 2 ⎝ ⎠

⎛ δ i + δ33i ⎞ i ⎟ = Δδi(γi) + ⎜δ22 − 11 2 ⎝ ⎠

(1)

where ζ represents the tilt angle to the helical axis to the bilayer normal, γi represents the phase angle that defines the location of the carbonyl carbon of the ith residue in the helix, and δi11, δi22, and δi33 represent the principal values of 13C CSA of the carbonyl carbon in the immobile state. This coordination system is illustrated in Figure S2. In eq 1, Δδi(γi) values oscillate as a function of γi, with an oscillation amplitude of (3/2) sin2 ζ, which is referred to as the “chemical shift oscillation”. As the overall structure of melittin bound to a DMPG bilayer is a rigid bent α-helix consisting of N- and C-terminal α-helices, the orientation of the melittin molecules bound to the membrane can be determined by calculating the root-mean-square deviations (RMSDs; eq 2) of the experimental CSAs, Δδobs, at G3, A4, and V5 for the Nterminus and L16, I17, and I20 for the C-terminus relative to the theoretical CSAs, Δδcal, using eq 1

RMSD(γ , ζ ) = RMSD(γ − 180°, ζ ) = RMSD(γ − 180°, −ζ ) = RMSD(γ , −ζ )

∑ {(Δδobs)i − (Δδcal)i }2 /n i=1

(4)

For the N- and C-terminal helices in the melittin−DMPG bilayer systems, the lowest RMSD values are given by

n

RMSD =

(3)

(γG3 , ζ )N = ( +71°, ±32°) or ( − 109°, ± 32°) and

(2)

(γL16 , ζ )C = ( +80°, ±30°) or (− 100°, ±30°)

and 1806

(5)


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alignment. Because basic residues (K21, R22, K23, R24) are located in one turn of the C-terminal helix, this turn should be located in the interfacial part of the lipid bilayer. Thus, by taking into account the large tilt angles relative to the bilayer normal for the N- and C-terminal α-helices, the N-terminal residue should be located inside the bilayer, because the transmembrane region should be shorter than the hydrophobic core thickness of the DMPG bilayer. This is defined as a pseudotransmembrane helix. It should be noted that the pseudotransmembrane alignment and topology of melittin bound to the DMPG bilayers is similar to the pseudotransmembrane alignment and topology of the melittin−DLPC and melittin−DPPC bilayers.30 In the case of melittin−DPPC bilayers with a thick core region, the N-terminal residues are located further inside the bilayer than in the case of melittin− DMPG bilayers. Orientation and Dynamic Structure, As Revealed by MD Simulation Studies. Figure 7a shows a snapshot

Figure 6. (A) Chemical shift oscillation pattern of melittin N- and Cterminal helices bound to DMPG bilayers. Gray, green, and blue lines indicate calculated chemical shift oscillations for G3, A4, and V5 in the N-terminus and L16, I17, and I20 in the C-terminus, respectively. (B) Schematic representation of the structure and orientation of melittin bound to DMPG bilayers. The Z axis is the axis of melittin about the bilayer normal (Z′) with the tilt angles (ζ = −32° for the N-terminal helix and +30° for the C-terminal helix). (C) Helix wheel phase angles are +71° with respect to G3 for the N-terminus and −100° with respect to L16 for the C-terminus.

In our previous study, we determined that (γG3, ζ)N = (+76°, −36°) and (γL16, ζ)C = (−82°, +25°) for melittin−DPPC bilayers. In this case, interatomic distances between [1-13C]V8 and [15N]L13 was measured to be 4.8 Å. This distance allowed to uniquely determine (γG3, ζ)N and (γL16, ζ)C values to show a larger kink angle. Because the labeled positions are the same as in the case of melittin−DMPG bilayer systems, it is appropriate to choose (γG3, ζ)N = (+71°, −32°) and (γL16, ζ)C = (−100°, +30°). The structure and orientation of melittin bound to the DMPG bilayers were analyzed in detail using the chemical shift oscillation patterns for the 13C Δδ values of the carbonyl carbons for the N- and C-terminal α-helices against the index number of residues (Figure 6A). The chemical shift oscillation pattern data clearly indicate that melittin forms N- and Cterminal α-helices with different tilt angles. In addition, melittin assumes a bent α-helical topology with a possible kink angle of 118° as estimated on the basis of the melittin−DPPC and melittin−DLPC bilayer systems30 under the assumption that the XN and XC axes are collinear. This assumption is justified in the cases of melittin−DPPC and melittin−DLPC bilayer systems,30 although small deviations from collinearity between the XN and XC axes cannot be ruled out. The structure and orientation of melittin bound to the DMPG bilayers as determined by NMR spectroscopy are shown in Figure 6B. Melittin molecules were inserted into the DMPG bilayer with a bent α-helical structure with tilt angles of 30° for the Cterminal helix and −32° for the N-terminal helix showing a possible kink angle of 118°, leading to a transmembrane

Figure 7. Snapshot illustration of melittin in the DMPG membrane bilayer after a 30 ns MD simulation (a). Water molecules are shown as “points”. The peptide backbone and residues G1 and K7 are shown as “cartoon” and “licorice” representations, respectively. For simplicity, lipid molecules and sodium and chloride ions are not shown. Illustration of the structure of the head and tail of a DMPG lipid molecule (b).

illustration of the melittin−DMPG system after a 30 ns MD simulation. Melittin assumed different tilt angles on the N- and C-terminal sides of the helices. Movement of melittin within the membrane is shown in Figure 8a. The position of the peptide relative to the membrane normal was analyzed over time for residue I2 in the N-terminal helix, Q25 in the Cterminal helix, and in the center of the melittin molecule. It can be seen that all three positions exhibited a similar movement in the membrane, that is, the entire melittin peptide moved in the C-terminal direction relative to the membrane normal, without any large change in the overall conformation of the peptide. Each position of the molecule moved rapidly at the beginning of the simulation and then fluctuated around the equilibrium value. Similarly, although the tilt angles of the peptide fluctuated, they did not change significantly during the simulation (Figure 8b). The tilt angles, ζ, of the N- and Cterminal helices averaged over the last 10 ns of the simulation were 35° (equivalent to −32°, which was determined by solidstate NMR) and 18°, respectively, which are in good agreement with −32° and 30°, respectively, as determined by solid-state 1807


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Figure 9. Changes in the interaction energy between the melittin (MLT) peptide and DMPG molecules (red), DMPG head groups (blue), and DMPG tail groups (green) over time (a). Changes in the interaction energy between the DMPG head group and G1 (red), K7 (blue), and the total positively charged residues in the C-terminus (green) of melittin (b).

DMPG head groups having negative net charge. Therefore, the interaction energy between positively charged residues in the melittin peptide and the negatively charged lipid head groups was analyzed, and the results are shown in Figure 9b. In particular, residues K21, R22, K23, and R24 in the C-terminus interacted strongly with the lipid head groups. As these four basic amino acids are sequential and make one turn of the αhelix, this region would interact strongly with the head groups of the surrounding membrane lipid molecules. As a result, the entire molecule is pulled up to the C-terminal direction. Therefore, the C-terminal side protrudes into the water region, whereas the N-terminal portion of the molecule is pulled up inside the core of the lipid bilayer. Melittin thus assumes a pseudotransmembrane alignment as shown in Figure 7a. On the N-terminal side of the molecule, in contrast, the positively charged residues, G1 and K7, also strongly interact with the lipid head groups, with almost equal interaction energies. The strong interaction of K7 is of particular interest because this hydrophilic residue is not located at the surface of the membrane but rather in the inner region of the bilayer. As shown in Figure 7a, the side chain of K7 is oriented toward the membrane interface region. Careful observations indicated that some of the water molecules are pulled into the membrane layer around the K7 side chain moiety. In other words, the side chain of K7 might interact directly with the water region, even though K7 is located within the membrane. As the C-terminal side binds tightly to the membrane in the region of the positively charged residues, the interactions of K7 and G1 on

Figure 8. Positions of I2 (blue), Q25 (green), and the center of the melittin (MLT) molecule (red) along the membrane normal over time (a). Change in the tilt angle, ζ, over time for the helices, measured by the angles between the CO group of G3 for the N-terminus (red), L16 for the C-terminus (blue), and the membrane normal (b). The helical wheel phase angle, γ, of the carbonyl carbons of G3 and L16 (c). The bold light green lines in (b) and (c) indicate the moving averages of the tilt angles and wheel phase angles, which were calculated every 100 steps.

NMR. The helical-wheel phase angle, γ, for the carbonyl carbon of the backbone chain over the course of the simulation is shown in Figure 8c. The average γ values over the last 10 ns of the simulation was 41° for G3 and −135° for L16, which are in good agreement with 71° for G3 and −100° for L16, as determined by solid-state NMR. Thus, the structure and orientation of membrane-bound melittin determined by MD simulation were in good agreement with those obtained experimentally by solid-state NMR analysis (Figure 6b). Interactions of Melittin with DMPG Membranes, As Revealed by MD Simulation Studies. To investigate how the orientation of the melittin molecule in the DMPG membranes is controlled, we analyzed the interactions between melittin and lipid molecules. Figure 9a shows the time course data for the interaction energy of the melittin peptide with the surrounding lipid molecules. Obviously, melittin interacts strongly with the DMPG molecules, with most of the interaction energy originating from the interaction with the 1808


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simulation by the interaction energy shown in Figure 10b. Similar phenomena were observed in the case of melittin in DLPC, DMPC, DPPC, and palmitoyloleoylphosphatidylcholine bilayers.30,37,38,54 The disruption of the membrane discussed above could be considered as the origin of the destruction of the membrane as observed in the experiment. The penetration of the water molecules, as illustrated in Figure 7a, was considered to be the result of disordering of the orientation of the DMPG molecule due to the interaction between K7 and the membrane.

the opposite side of the helix would lead to tilting of the Nterminal helical axis to the membrane normal. Figure 9b shows the strong interaction between K7 and the surrounding lipid molecules over the entire MD simulation time, with the exception of the beginning stage. Figure 10a

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CONCLUSIONS P NMR spectra of DMPG bilayers containing melittin demonstrated disruption of the membrane at temperatures below Tc. The melittin−DMPG system exhibited weaker membrane disruption than the melittin−DMPC, melittin− DLPC, and melittin−DPPC systems. The chemical shift oscillation analyses using 13C solid-state NMR spectroscopy indicate that melittin inserts into the DMPG bilayer in a pseudotransmembrane alignment, assuming a bent α-helical structure with a tilt angle of −32° for the N-terminal helix and 30° for the C-terminal helix to the bilayer normal and a probable kink angle of 118°. This structure is almost identical to that of melittin associated with neutral DMPC, DLPC, and DPPC bilayers. MD simulation studies indicated that the basic amino acid residues K21, R22, K23, and R24 in the C-terminus of melittin interact strongly with the hydrophilic lipid head groups, such that the C-terminal moiety of the inserted melittin protruded from the surface of the DMPG lipid bilayers. However, the basic amino acid residue K7 is located slightly within the lipid core region and interacts with one DMPG molecule to disturb the lower surface of the lipid bilayer, which is associated with membrane disruption. Interaction of K7 with an acidic DMPG molecule may also explain the weaker disruption ability of melittin to DMPG than to DMPC because interaction of K7 with acidic DMPG is stronger than with neutral DMPC, which reduces the disruption ability. 31

Figure 10. Snapshot illustration of melittin and lipid molecules in a 28 ns MD simulation (a). The interaction between a selected DMPG molecule and the side chain of melittin residue K7 is shown in the inset. Changes in the interaction energy between K7 and DMPG (b). The interaction energy was calculated between residue K7 and two phosphate oxygens of the DMPG molecule. Comparison of changes in the position of the DMPG phosphorus atom selected above and the average position of the phosphorus atoms in the lower layer of the lipids (c).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b11207. Analysis of dynamic structure of melittin bound to membrane (text); temperature variation in 31P chemical shift anisotropy (Table S1); micrographs illustrating lysis in melittin−DMPG bilayer systems (Figure S1); coordination system of the rotating α-helix (Figure S2); coordinate conversion using Euler rotation angles for description of the rotational motion of α-helical melittin bound to the membrane (Figure S3) (PDF)

shows a snapshot picture of melittin surrounded by lipid molecules at the N-terminus. It was found that melittin interacts with a neighboring membrane molecule at the K7 residue. The inset illustrates the electrostatic interaction between the NH3+ group of the melittin K7 side chain and the negatively charged phosphate oxygen of a DMPG molecule. The interaction energy between K7 and the negatively charged phosphate oxygens of a particular DMPG molecule is shown in Figure 10b. This selected lipid molecule began to interact with melittin after 25 ns, indicating that the lipid molecule was alternately exchanged to interact with residue K7 of melittin. This interaction pulled up the lipid molecule inside the bilayer, thus disrupting the membrane alignment. Disruption of the membrane alignment was evaluated based on the position of the phosphorus atoms in the lower layer of the membrane. The results showed that the average position of the phosphorus atoms in the lower layer of the membrane was almost constant over the entire simulation after 5 ns (Figure 10c), indicating that the membrane, as a whole, maintained a well-ordered structure over the entire simulation. In contrast, the position of the phosphorus atom of the selected DMPG molecule was found to be pulled into the membrane after the 25 ns

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (K.U.). *E-mail: [email protected] (A.N.). ORCID

Izuru Kawamura: 0000-0002-8163-9695 Akira Naito: 0000-0003-2443-6135 Notes

The authors declare no competing financial interest. 1809


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(19) Dawson, C. R.; Drake, A. F.; Helliwell, J.; Hider, R. C. The interaction of bee melittin with lipid bilayer membranes. Biochim. Biophys. Acta 1978, 510, 75−86. (20) Knöppel, E.; Eisenberg, D.; Wickner, W. Interaction of melittin, a protein model, with detergents. Biochemistry 1979, 18, 4177−4181. (21) Inagaki, F.; Shimada, I.; Kawaguchi, K.; Hirano, M.; Terasawa, I.; Ikura, T.; Go, N. Structure of melittin bound to perdeuterated dodecylphosphocholine micelles as studied by two-dimensional NMR and distance geometry calculation. Biochemistry 1989, 28, 5985−5991. (22) Okada, A.; Wakamatsu, K.; Miyazawa, T.; Higashijima, T. Vesicle bound conformation of melittin: transferred nuclear Overhauser enhancement analysis in the presence of perdeuterated phosphatidylcholine vesicles. Biochemistry 1994, 33, 9438−9446. (23) Citra, M. J.; Axelsen, P. H. Determination of molecular order in supported lipid membrane by internal reflection Fourier transform infrared spectroscopy. Biophys. J. 1996, 71, 1796−1805. (24) Altenbach, C.; Froncisz, W.; Hyde, J. S.; Hubbell, W. L. Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation electron paramagnetic resonance. Biophys. J. 1989, 56, 1183−1191. (25) Stanislawski, B.; Ruterjans, H. 13C-NMR investigation of the insertion of the bee venom melittin into lecithin vesicles. Eur. Biophys. J. 1987, 15, 1−12. (26) Dempsey, C. E.; Butler, G. S. Helical structure and orientation of melittin in dispersed phospholipid membranes from amide exchange analysis in situ. Biochemistry 1992, 31, 11973−11977. (27) Hristova, K.; Dempsey, C. E.; White, S. H. Structure, location, and lipid perturbation of melittin at the membrane interface. Biophys. J. 2001, 80, 801−811. (28) Smith, R.; Separovic, F.; Milne, T. J.; Whittaker, A.; Bennett, F. M.; Cornell, B. A.; Makriyannis, A. Structure and orientation of the pore forming peptide, melittin, in lipid bilayers. J. Mol. Biol. 1994, 241, 456−466. (29) Naito, A.; Nagao, T.; Norisada, K.; Mizuno, T.; Tuzi, S.; Saitô, H. Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state 31P and 13C NMR spectroscopy. Biophys. J. 2000, 78, 2405−2417. (30) Toraya, S.; Nishimura, K.; Naito, A. Dynamic structure of vesicle-bound melittin in a variety of lipid chain lengths by solid state NMR. Biophys. J. 2004, 87, 3323−3335. (31) Lam, Y.-H.; Wassall, S. R.; Morton, C. J.; Smith, R.; Separovic, F. Solid-state NMR structure determination of melittin in a lipid environment. Biophys. J. 2001, 81, 2752−2761. (32) Frey, S.; Tamm, L. K. Orientation of melittin in phospholipids bilayers. A polarized attenuated total reflection infrared study. Biophys. J. 1991, 60, 922−930. (33) Uezono, T.; Toraya, S.; Obata, M.; Nishimura, K.; Tuzi, S.; Saitô, H.; Naito, A. Structure and orientation of dynorphin bound to lipid bilayers by 13C solid-state NMR. J. Mol. Struct. 2005, 749, 13−19. (34) Toraya, S.; Javkhlantugs, N.; Mishima, D.; Nishimura, K.; Ueda, K.; Naito, A. Dynamic structure of bonbolitin II bound to lipid bilayers as revealed by solid-state NMR and Molecular dynamics simulation. Biophys. J. 2010, 99, 3282−3289. (35) Tsutsumi, A.; Javkhlantugs, N.; Kira, A.; Umeyama, M.; Kawamura, I.; Nishimura, K.; Ueda, K.; Naito, A. Structure and orientation of bovine lactoferrampin in the mimetic bacterial membrane as revealed by solid-state NMR and molecular dynamics simulation. Biophys. J. 2012, 103, 1735−1743. (36) Nagao, T.; Mishima, D.; Javkhlantugs, N.; Wang, J.; Ishioka, D.; Yokota, K.; Norisada, K.; Kawamura, I.; Ueda, K.; Naito, A. Structure and orientation of antibiotic peptide alamethicin phospholipid bilayers as revealed by chemical shift oscillation analysis of solid state nuclear magnetic resonance and molecular dynamics simulation. Biochim. Biophys. Acta 2015, 1848, 2789−2798. (37) Bernèche, S.; Nina, M.; Roux, B. Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane. Biophys. J. 1998, 75, 1603−1618.

ACKNOWLEDGMENTS This work was supported by grants-in-aid for Scientific Research in an Innovative Area (JP16H00756 and JP16H00828), and by a grant-in-aid for Scientific Research (C) (JP15K06963) and Research (B) (JP15H04336) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors (N.J. and K.U.) acknowledge a Mongolia−Japan Education Engineering Development (MJEED) cooperative (J11B16) grant from the Japan International Cooperation Agency and the Ministry of Education, Culture and Science, and Sports of Mongolia for supporting a portion of the MD simulation work.

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REFERENCES

(1) Habermann, E.; Jentsch, J. Seequenzanalyse des melittin aus den tryptischen und peptischen spaltücken. Hoppe-Seyler’s Z. Physiol. Chem. 1967, 348, 37−50. (2) Habermann, E. Bee and wasp venoms. Science 1972, 177, 314− 322. (3) Sessa, G.; Freer, J. H.; Colaccico, G.; Weissmann, G. Interaction of a lytic polypeptide, melittin, with lipid membrane systems. J. Biol. Chem. 1969, 244, 3575−3582. (4) Mollay, C.; Kreil, G. Enhancement of bee venom phospholipase A2 activity by melittin, direct lytic factor from cobra venom and polymyxin B. FEBS Lett. 1974, 46, 141−144. (5) Mollay, C.; Kreil, G.; Berger, H. Action of phopholipases on the cytoplasmic membrane of Escherichia coli. Biochim. Biophys. Acta 1976, 426, 317−324. (6) Morgan, C. G.; Williamson, H.; Fuller, S.; Hudson, B. Melittin induces fusion of unilamellar phospholipid vesicles. Biochim. Biophys. Acta 1983, 732, 668−674. (7) Eytan, G. D.; Almary, T. Melittin-induced fusion of acidic liposomes. FEBS Lett. 1983, 156, 29−32. (8) Dufourcq, J.; Faucon, J.-F.; Fourche, G.; Dasseux, J.-L.; Le Maire, M.; Gulik-Krzywicki, T. Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles. Biochim. Biophys. Acta 1986, 859, 33−48. (9) Faucon, J.-F.; Bonmatin, J.-M.; Dufourcq, J.; Dufourc, E. J. Acylchain-length dependence in the stability of melittin-phosphatidylcholine complexes. A. light scattering and 31P-NMR study. Biochim. Biophys. Acta 1995, 1234, 235−243. (10) Tosteson, M. T.; Tosteson, D. C. Melittin forms channels in lipid bilayers. Biophys. J. 1981, 36, 109−116. (11) Kempf, C.; Klausner, R. D.; Weinstein, J. N.; Van Renswoude, J.; Pincus, M.; Blumenthal, R. Voltage-dependent trans-bilayer orientation of melittin. J. Biol. Chem. 1982, 257, 2469−2476. (12) Talbot, J. C.; Dufourcq, I.; de Bony, J.; Faucon, J. F.; Lussan, C. Conformational change and self-association of monomeric melittin. FEBS Lett. 1979, 102, 191−193. (13) Bello, J.; Bello, H. R.; Granados, E. Conformation and aggregation of melittin: dependence on pH and concentration. Biochemistry 1982, 21, 461−465. (14) Quay, S. C.; Condie, C. C. Conformational studies of aqueous melittin: thermodynamics parameters of the monomer-tetramer self association reaction. Biochemistry 1983, 22, 695−700. (15) Anderson, D.; Terwilliger, T. C.; Wickner, W.; Eisenberg, D. Melittin forms crystals which are suitable for high resolution x-ray structural analysis and which revel a molecular twofold axis of symmetry. J. Biol. Chem. 1980, 255, 2578−2582. (16) Terwilliger, T. C.; Eisenberg, D. The structure of melittin. J. Biol. Chem. 1982, 257, 6010−6015. (17) Terwilliger, T. C.; Weissman, L.; Eisenberg, D. The structure of melittin in the form I crystal and its implication for melittin’s lytic and surface activity. Biophys. J. 1982, 37, 353−361. (18) Bazzo, R.; Tappin, M. J.; Pastore, A.; Harvey, T. S.; Carver, J. A.; Campbell, I. D. The structure of melittin. A 1H-NMR study in methanol. Eur. J. Biochem. 1988, 173, 139−146. 1810


Article

The Journal of Physical Chemistry B (38) Bachar, M.; Becker, O. M. Protein-induced membrane disruption: a molecular dynamics study of melittin in a dipalmitoylphosphatidylcholine bilayers. Biophys. J. 2000, 78, 1359−1375. (39) Javkhlantugs, N.; Naito, A.; Ueda, K. Molecular dynamics simulation of bombolitin II in the dipalmitoylphosphatidylcholine membrane bilayer. Biophys. J. 2011, 101, 1212−1220. (40) Paquet, A. Introduction of 9-fluorenylmethyloxycarbonyl, trichloroethoxycarbonyl, and benzyloxycarbonyl amine protecting group into unprotected hydroxylamine acids using succinimidyl carbonate. Can. J. Chem. 1982, 60, 976−980. (41) King, D. S.; Fields, C. G.; Fields, G. B. A cleavage method which minimizes side reaction following Fmoc solid phase peptide synthesis. Int. J. Pept. Protein Res. 1990, 36, 255−266. (42) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 1983, 4, 187−217. (43) Mackerell, A. D.; Bashford, D.; Bellott, D.; Dunbrack, R. L.; Evanseck, R. L.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; JosephMcCarthy, D.; et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 1998, 102, 3586−3616. (44) Klauda, J. B.; Venable, R. M.; Freites, J. A.; O’Connor, J. W.; Tobias, D. J.; Mondragon-Ramirez, C.; Vorobyov, I.; MacKerell, A. D.; Pastor, R. W. Updata of the CHARMM all-atom additive force field for lipids: Validation on six lipid types. J. Phys. Chem. B 2010, 114, 7830− 7843. (45) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. M.; Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926−935. (46) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. Numerical integration of the cartesian equations of motion of a system with constrains: molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327−341. (47) Ewald, P. D. The calculation of optical and electrostatic lattice potentials [Die berechnung optischer und elektrostatischer gitterpotentiale]. Ann. Phys. 1921, 369, 253−287. (48) Nosé, S. A unified formulation of the constant temperature molecular dynamics methods. J. Chem. Phys. 1984, 81, 511−519. (49) Hoover, W. G. Canonical dynamics: Equilibrium phase-space distributions. Phys. Rev. A 1985, 31, 1695−1697. (50) Humphrey, W.; Dalke, A.; Schulten, K. VMD-Visual molecular dynamics. J. Mol. Graphics 1996, 14, 33−38. (51) Toraya, S.; Nagao, T.; Norisada, K.; Tuzi, S.; Saitô, H.; Izumi, S.; Naito, A. Morphological behavior of lipid bilayer induced by melittin near the phase transition temperature. Biophys. J. 2005, 89, 3214− 3222. (52) Saitô, H. Conformation-dependent 13C chemical shifts: a new means of conformation characterization as obtained by high-resolution solid-state NMR. Magn. Reson. Chem. 1986, 24, 835−852. (53) Saitô, H.; Ando, I. High-resolution solid-state NMR studies of synthetic and biological macromolecules. Annu. Rep. NMR Spectrosc. 1989, 21, 209−290. (54) Irudayam, S. J.; Berkowitz, M. L. Binding and reorientation of melittin in a POPC bilayer: Computer simulations. Biochim. Biophys. Acta 2012, 1818, 2975−2981.

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Dynamic Structure and Orientation of Melittin Bound to Acidic Lipid

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