Dynamical and Phase Behavior of a Phospholipid Membrane Altered

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Letter

Dynamical and Phase Behavior of a Phospholipid Membrane Altered by an Antimicrobial Peptide at Low Concentration Veerendra Kumar Sharma, Eugene Mamontov, Madhusudan Tyagi, Shuo Qian, Durgesh K. Rai, and Volker S. Urban J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b01006 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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The Journal of Physical Chemistry Letters

Dynamical and Phase Behavior of a Phospholipid Membrane Altered by an Antimicrobial Peptide at Low Concentration V.K.Sharma*1,2, E. Mamontov3,M. Tyagi4,5, S. Qian1, D. K. Rai1and V.S. Urban1

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Biology and Soft Matter Division, Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

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Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400085, India

Chemical and Engineering Materials Division, Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

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National Institute of Standards and Technology Center for Neutron Research, Gaithersburg, Maryland 20899, USA 5

Department of Materials Science and Engineering, University of Maryland, College Park, Maryland 20742, USA

* Corresponding Author(s): V. K. Sharma, E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment

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Abstract The mechanism of action of antimicrobial peptides is traditionally attributed to formation of pores in the lipid cell membranes of pathogens, which requires a substantial peptide to lipid ratio. However, using incoherent neutron scattering, we show that even at a concentration too low for pore formation, an archetypal antimicrobial peptide, melittin, disrupts the regular phase behavior of the microscopic dynamics in a phospholipid membrane, dimyristoylphosphatidylcholine (DMPC). At the same time, another antimicrobial peptide, alamethicin, does not exert similar effect on the DMPC microscopic dynamics. The melittin-altered lateral motion of DMPC at physiological temperature no longer resembles the fluid-phase behavior characteristic of functional membranes of the living cells. The disruptive effect demonstrated by melittin even at low concentrations reveals a new mechanism of antimicrobial action relevant in more realistic scenarios, when peptide concentration is not as high as would be required for pore formation, which may facilitate treatment with antimicrobial peptides.

TOC Graphics

Keywords: Antimicrobial peptides, Melittin, Alamethicin, Phosphocholine membrane, Dynamics, Neutron scattering, Membrane-peptide interactions


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Antimicrobial peptides (AMPs) play a key role in immunological defense against invasive microorganisms in all forms of life, from bacteria to plants, insects, fish, and mammals.1,2 Due to their broad spectrum of activity, lower levels of bacterial resistance, and the speed of their action on pathogens, AMPs have been recognized as a potential candidate for treatment of antibioticresistant bacterial infections, septic shock, cancer cells, etc.3,4 The main target of AMPs is the cell membrane, which is a complex heterogeneous mixture of lipids, proteins, and other small molecules.5Amphipathicity, charge, and size of AMPs favor the attachment or insertion of these peptides into the membrane bilayers. The AMPs-membrane interaction depends on various parameters, such as membrane composition, properties of the lipid and peptide, physical state of the bilayer, peptide to lipid ratio, etc.1,6 Because it has been shown1,6that AMPs action is the result of direct interaction with the cell membrane rather than possession of specific membrane protein receptors, a model membrane system composed of phospholipids may be well suited for elucidating the fundamental mechanism of the AMPs action on pathogen cell membranes. In this work, we compare the effects exerted by prominent AMPs, melittin and alamethicin, on the microscopic dynamics and phase behavior of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane. We discover that even at a low concentration, insufficient to induce pores in the membrane, the highly charged melittin completely alters the phase behavior of DMPC microscopic dynamics. Thus, low amounts of melittin disrupt the fluid-like diffusion dynamics of the membrane lipids, which is associated with the function of the living cell membranes (including those of pathogens) at physiological temperatures. This suggests a viable mechanism of antimicrobial action by melittin that does not require high melittin concentrations, which would be needed to create pores in a pathogen’s membrane. Furthermore, it opens a broad question as to whether other AMPs might be capable of acting on pathogen’s membrane in a similar manner, at concentrations that are too low to induce the transmembrane pores. Alamethicin is a small 20-amino acid antibiotic peptide known to induce a voltagedependent conductance in the lipid bilayer.7,8 At neutral pH, it carries a single negative charge. Melittin is a small linear amphipathic peptide consisting of 26 amino acids, of which residues 1– 20 (amino-terminal region) are mostly hydrophobic, while residues 21–26 (carboxy-terminal region) are hydrophilic. At neutral pH, it carries a net charge of +5.9,10 It readily associates with other amphiphilic structures, such as lipid bilayers. AMPs exist in solution with random secondary structures. However, in contact with a membrane, both alamethicin and melittin


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assume an alpha helical secondary structure.2At low concentrations in the membrane, the helical peptide adsorbs into the polar region, mainly lying parallel to the plane of the bilayer. At high concentration, the peptide changes its orientation and starts creating transmembrane pores.11-15 Alamethicin is known to form barrel-stave pores, in which peptides associate to form a bundle with a central lumen, like a barrel made of helical peptides as staves.14In this model, the pore is lined entirely by helical peptides. On the other hand, melittin is known to form toroidal pores, in which the lipid monolayer continuously bends from the top leaflet to the bottom leaflet through a toroidal hole, so the pore is lined by both the lipid head groups and peptide monomers.15In this model, peptides are always associated with the lipid head groups, even when they are oriented perpendicular to the bilayer. Stable transmembrane pores with well-defined density are formed only when the peptide concentration exceeds the critical peptide to lipid ratio.11Various perturbations in the membrane morphology induced by melittin and alamethicin, such as bilayer thinning/thickening, pore formation, promotion of nonlamellar lipid structures, or detergent-like action, have been reported.6,11-21At peptide concentrations lower than the threshold concentration required for the formation of stable transmembrane pores, a secondary stressful effect on the membrane, resulting from a disruption of the lipid distribution between the inner and outer leaflets of the bilayer, has been suggested in a recent small angle neutron scattering (SANS) study.17However, the effect of alamethicin and melittin on the dynamical behavior of lipid bilayer have not been widely studied. Quasielastic neutron scattering (QENS) is one of the best technique suitable for studying microscopic dynamics of self-assembled aggregates on a pico- to nanosecond timescale and on a length scale from Angstroms to tens of nanometers.22-38 Recently, we have used QENS to investigate the solid gel to fluid phase transition in DMPC unilamellar vesicles (ULVs)32and the mitigating effect of cholesterol on the peptide-induced destabilization of DMPC.33Elastic incoherent neutron scattering readily detects phase transitions in a phosphocholine membrane, which include a pre-transition (to solid gel-ripple phase) below the chain-melting main phase transition of lipid membranes.34For DMPC membrane, the pre-transition and main transitions are observed at ~287 and 297 K, respectively. Likewise, calorimetry results from the literature support the transitions in pure DMPC at these temperatures, yet indicate broadening of the peak corresponding to the main phase transition in presence of melittin39-40. The phase state of the bilayer affects its interaction with AMPs.6,20,33,41-43Therefore, we need to investigate the role of


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the physical state of the bilayer in the interaction of melittin and alamethicin with DMPC membrane. To make a meaningful comparison between the two AMPs, all experimental conditions, such as peptide to lipid ratio, lipid composition, temperature, etc., are kept uniform between the measurements with melittin and alamethicin. For neutron scattering measurements, we have deliberately selected a low AMP concentration of 0.5 mol %. Numerous studies have shown that melittin and alamethicin do not form membrane pores at this concentration with most lipid compositions.12,14,15,17Nevertheless, we need to rule out a possibility that the dynamics and phase behavior effects observed on DMPC membrane could be due to pore formation by peptide, especially melittin, which exhibits much stronger effect on the bilayer than alamethicin. To this end, we performed neutron in-plane scattering and oriented circular dichroism experiments on multi-lamellar bilayers with melittin. The neutron in-plane scattering makes effective use of the scattering contrast between H2O and D2O to detect structural features in the plane of the multilamellar samples. We compared three samples: pure DMPC, DMPC with 0.5 mol % melittin (i.e., Melittin/DMPC=1/200), and DMPC with 4 mol % melittin (i.e. Melittin/DMPC=1/25). All samples were hydrated with D2O. If there is a membrane pore present in the bilayer plane, the D2O in the pore column exhibits a scattering contrast with the surrounding hydrogenated lipidpeptide complex. Neutron in-plane scattering is able to detect membrane pores that diffuse as disks in a two-dimensional membrane plane. As shown in Figure 1a, the sample with DMPC showed a sharp peak around 0.1 Å-1. This is a lamellar diffraction peak corresponding to a dspacing of 62 Å, indicating a well hydrated sample. This peak is caused by oily streak defects44, and it can be seen in all other samples, sometimes on the top of other scattering peaks. DMPC with 4 mol % melittin shows an additional broad peak around 0.07 Å-1 resulting from the inplane scattering of D2O columns, which indicates the presence of membrane pores.44The small lamellar peak on the right shoulder of this in-plane peak can be eliminated by subtracting a Gaussian positioned at 0.099 Å-1. Then the in-plane scattering peak (inset of Figure 1(a)) can be fitted well assuming membrane pores of density ~0.65 in the plane, a pore-to-pore distance ~102 Å, and a D2O column (that is, pore radius) of 22 Å. Compared to 4 mol % melittin, the sample of interest to us, DMPC with 0.5 mol % melittin does not show in-plane scattering peak, but only a relatively small lamellar peak. This demonstrates that the melittin concentration we used in QENS experiments was not sufficient to form stable pores.


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Figure 1 (a) In-plane neutron scattering pattern from a sandwiched sample of fully hydrated DMPC with D2O at different concentrations of melittin: 0 (black square), 0.5 mol % (red circle) and 4 mol % (blue triangle) at 100% RH D2O. Inset: data for DMPC with 4 mol % melittin after removing shoulder peak on the right. Solid line is the fits as per the model discussed in the text. (b) Oriented circular dichroism (OCD) spectra of the same samples (i.e., DMPC with 0.5 and 4 mol % melittin) at 100 % RH, on which in-plane scattering measurements have been performed. (c) SANS data on DMPC vesicles with and without 0.5 mol % melittin and the fits obtained using Core 3-shell model. (d) Bilayer thickness from SANS fits as a function of temperature for DMPC with and without 0.5 mol % melittin.

The same samples were then measured by oriented circular dichroism (OCD) to detect the orientation of melittin. The orientation of peptide in the lipid bilayer is an important indicator of the mode of peptide association with the membrane, providing clues on how peptide disturbs the bilayer. Previous studies11-13 have shown that at low concentration peptide is lying parallel to


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the bilayer, with little expectation of membrane pore formation (S-state). When a membrane pore forms, usually at peptide concentrations exceeding the critical concentration, a significant fraction of peptide is found perpendicular to the bilayer (I-state), inserting into the membrane.12 The OCD spectrum from 4 mol % melittin (Figure. 1b) resembles an I-state, indicating peptide insertion and pore formation. Contrary to that, 0.5 mol % melittin exhibits OCD spectrum clearly different in shape, showing a majority of peptide not inserted, but in S-state instead. From the neutron in-plane and OCD experiments, we unambiguously verified that there are no membrane pores at the low melittin concentration that we used for the dynamics experiments, thus ruling out a possibility of pore formation as the mechanism of melittin-induced membrane disturbance reported here. To characterize the structural behavior of the lipid bilayer, SANS was performed on DMPC ULV samples with and without 0.5 mol % melittin at 280, 290, 300 and 310K. The data, along with the analysis results obtained using Core 3-Shell model, are shown in Figure 1(c). The resulting bilayer thicknesses are plotted in Figure 1(d), which shows that addition of melittin has modest effect on the bilayer thickness. In the ordered phase (280 and 290K), while addition of melittin decreases the bilayer thickness, in the fluid phase (310K), it leads to slight increase in the bilayer thickness relative to pure DMPC. The free volume, which is inversely proportional to the bilayer thickness, thus shows much smaller variation with temperature for DMPC in presence of melittin compared to pure DMPC. Lipid dynamics associated with the phase behaviour in a sample can be investigated by measuring the elastic intensity scan as a function of temperature, when a step-like increase/decrease in the elastic intensity indicates a phase transition. In elastic scan, intensity is measured center at zero energy transfer and can be regarded elastic if the scattering takes place within the energy resolution of the instrument. To observe the low-enthalpy solid gel-ripple phase pre-transition, along with the main phase transition, elastic intensity scans have been carried out using a very slow heating/cooling rate of 0.1 K/min at a sensitive neutron spectrometer, HFBS45. Molecular motions slower than ~1 ns contribute to the elastic intensity measured at HFBS (∆EHFBS~0.8 µeV). Figure 2 shows the elastic intensity averaged over scattering angles corresponding to a Q range of 0.36 to 1.75 Å-1 for DMPC vesicles in the presence and absence of 0.5 mol % alamethicin/melittin. In the cooling cycle of DMPC (Figure 2a), the elastic scan data indicate a sudden jump at ~297 K as well as a small kink at ~286 K.


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Likewise, in the heating cycle of DMPC, two steps are clearly visible at ~288 and 297 K. These temperatures match well the temperature of the pre- and main phase transition of DMPC bilayer46. A small temperature hysteresis exists mainly for the pre-transition, which appears more pronounced in the heating cycle. Figure 2b shows the elastic scan data for DMPC vesicles with 0.5 mol % alamethicin. In this case, no pre-transition is observed in the cooling cycle, and only a very weak signature of pre-transition could be seen at ~288 K in the heating cycle. Likewise, the main phase transition, although always evident, is somewhat better defined in the heating cycle compared to the cooling cycle. Thus, inclusion of 0.5 mol % alamethicin suppresses the pretransition in DMPC. Figure 2c shows the elastic scan data for DMPC vesicles with 0.5 mol % melittin. It is evident that neither pre- nor main phase transition can be observed in this case. Thus, inclusion of 0.5 mol % melittin completely disrupts the regular phase behavior of the microscopic dynamics in a DMPC membrane such that no signs of pre- and main phase transitions are observed. The main phase transition is known to be strongly cooperative in character.36 Therefore, our measurement suggests that a single melittin molecule can prevent about a hundred of DMPC molecules from participating in the transition. This means that a single melittin molecule perturbs an entire cooperative group of phospholipids in such a way that their cooperativity is suppressed. Since this cannot be accounted only by local perturbations around the melittin, there must be long range effects of the incorporated melittin beyond its immediate neighborhood. It is instructive to compare the amplitude of elastic intensity of DMPC vesicles in the presence and absence of alamethicin/melittin. In the fluid phase (i.e., at temperatures above 297 K), we found that inclusion of 0.5 mol % alamethicin increases elastic intensity with respect to pure DMPC vesicles, whereas in the gel phase (i.e. temperature below 286 K) the intensity remains almost same. On the other hand, inclusion of 0.5 mol % melittin leads to the significantly decreased elastic intensity below the main transition temperature, whereas above the transition temperature the reverse trend is observed. At the very minimum, these observations suggest that addition of 0.5 mol % melittin makes the DMPC structure relatively less ordered at lower temperatures and relatively less disordered (that is, less fluid) at high temperatures. Moreover, the featureless character of the elastic intensity scans of DMPC with 0.5 mol % melittin demonstrates that, from the standpoint of the lipid microscopic dynamics, a single membrane phase exists through the entire temperature range of the current experiment.


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Figure 2 Q-averaged elastic intensity scans data for 0.1 M DMPC vesicles: (a) neat, (b) with 0.5 mol % of alamethicin, (c) with 0.5 mol % of melittin in heating and cooling cycles. Heating/cooling is performed with a rate of 0.1K/min. For clarity, all elastic scans in the heating cycles are shifted upward uniformly with respect to the elastic scans in the cooling cycles. Solid ines are drawn as guides to the eye. Error bars throughout the text represent one standard deviation.

This idea can be further tested through the analysis of dynamic QENS data. The characteristics of BASIS47 make it suitable for studying both lateral (on the nanosecond time scale) and internal motions (on the picosecond time scale) of lipid bilayer.32-34 QENS experiments have been carried out each vesicles solutions and solvent (D2O). Spectra from vesicles solution and D2O were first normalized to the incident beam monitor, and then the final scattered intensity from the vesicles, Ives(Q,ω), was obtained using the following relationship:

I ves ( Q, ω ) = I solution ( Q, ω ) − φ I D 2O ( Q, ω )

(1)

where φ is the volume fraction of DMPC. The typical spectra obtained at a representative Q =1.1 Å-1 are shown in Figure 3 at all three measured temperatures. The instrument resolution is also shown by the solid line in the upper left panel. For the purpose of comparison, the spectra are normalized to the peak amplitudes at each temperature. In agreement with the elastic intensity scans, at 310 K, where pure DMPC vesicles are in the fluid phase, addition of alamethicin and melittin leads to decreased signal broadening compared to pure vesicles, DMPC


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with melittin being the slowest. On the other hand, at 280 K and 293 K, where pure DMPC vesicles are in the solid gel and ripple phase, inclusion of melittin leads to increased signal broadening compared to pure vesicles, while the inclusion of alamethicin has little visible effect. The model scattering law for the vesicles can be expressed as23,32-34

Sves ( Q, ω ) = Slat ( Q, ω ) ⊗ Sint ( Q, ω )

(2)

where Slat(Q,ω) and Sint(Q,ω) correspond to the scattering functions describing the lateral and internal motions of the lipid molecules, respectively. The lateral and internal motions are well characterised by continuous diffusion29,30,32 and localized processes,23,32 respectively. Therefore, the overall scattering law for vesicles can be written as S ves ( Q, ω ) =  A ( Q ) Llat ( Γ lat , ω ) + (1 − A ( Q ) ) Ltot (Γ lat + Γ int , ω ) 

(3)

where Γlat and Γint are the half width at half maximum (HWHM’s) of the Lorentzian corresponding to the lateral and internal motion of the lipid molecules, respectively, and A(Q) is Elastic Incoherent Structure Factor (EISF) for the internal motion.22,23,32-34Eq. (3) was convolved with the instrumental resolution function, and the parameters A(Q),Γlat and Γint were determined by a least squares fit of the measured spectra. The data were fitted using DAVE package.48The observed QENS spectra for DMPC vesicles in the presence and absence of alamethicin/melittin are described well by the model scattering function (Eq. (3)) at all the measured temperature at all Q values. Typical fitting is shown in the lower right panel of Fig. 3. To gain more insight into the two dynamical processes, the parameters obtained from the fit are analyzed as a function of Q. The lateral motion is of the most interest to us since it plays an important role in many physiologically relevant membrane processes, such as protein-protein interaction, signalling and energy transduction pathways, etc. It is evident from Figure 4 that Γlat varies linearly with Q2 at all three temperatures, indicating continuous diffusion described by a simple Brownian motion using Fick’s law, HWHM=DlatQ2, as shown by the lines in Figure 4. Here Dlat is the diffusion coefficient for the lateral motion of the lipid molecules obtained from the slope of the straight line fit, as given in Table I. At 280 K, where pure DMPC vesicles are in the solid gel phase, the lateral diffusion coefficient of DMPC is found to be (0.7 ± 0.1) × 10-7 cm2/sec, which remains almost the same after inclusion of 0.5 mol % alamethicin. However, in the presence of 0.5 mol % melittin, the


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Figure 3 Typical QENS spectra measured at Q = 1.1 Å-1 for DMPC membrane, neat (solid black squares) and with 0.5 mol % alamethicin (solid red circles) or 0.5 mol % melittin (solid blue triangles) at three measured temperatures of 280, 293, and 310 K. The instrument resolution is shown by the solid line in the upper left panel. The contribution of the solvent (D2O) has been subtracted, and the resultant spectra are normalized to the peak amplitudes. Typical fitted QENS spectra for DMPC membrane with 0.5 mol % melittin measured at Q = 1.1 Å-1 assuming the model scattering function given by Eq. (3) is shown in the right bottom panel. lateral diffusion coefficient for DMPC lipid increases to (1.2 ± 0.1) × 10-7 cm2/sec. Similar results are observed at 293 K, where the inclusion of alamethicin does not affect the lateral diffusion significantly, whereas melittin increases it. In contrast to these results, at 310 K, where pure DMPC vesicles are in the fluid phase, both AMPs slow down the lateral diffusion. At 310 K, the lateral diffusion coefficient for DMPC vesicles is (5.0


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±0.2) × 10-7 cm2/sec, which is reduced to (3.9 ± 0.1) × 10-7 cm2/sec and (3.3 ± 0.1)× 10-7 cm2/sec with addition of alamethicin and melittin, respectively. Melittin acts as a stronger stiffening agent at 310 K, hindering the lateral diffusion more efficiently in comparison with alamethicin. At 280 K and 293 K, melittin enhances the dynamics, while alamethicin has no significant effects on the lateral motion.

Figure 4 Variation of the half width at half maximum of the Lorentzian corresponding to the lateral motion, Γlat, with Q2 for DMPC membrane, neat (circles) and with 0.5 mol% alamethicin (squares) or melittin (triangles) at all three measured temperatures of 280 K (filled), 293 K (halffilled) and 310 K (open). It is evident that for all samples, at each measured temperature, Γlat follows continuous diffusion model. The solid, dashed, and dashed-dotted lines are the fits with Fick’s law of diffusion at 280, 293, and 310K, respectively. Inset: Arrhenius plot of Dlat as obtained from the slope for neat DMPC membrane (circles) and DMPC in the presence of 0.5 mol % alamethicin (squares) or melittin (triangles).


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Table I. Lateral diffusion coefficient of DMPC in the bilayer in the presence and absence of melittin/alamethicin. T (K)

Lateral diffusion coefficient (Dlat) (×10-7 cm2/s) DMPC

DMPC +Alamethicin

DMPC+Melittin

280 K

0.7 ± 0.1

0.7± 0.1

1.2 ± 0.1

293 K

1.4 ± 0.1

1.3 ± 0.1

1.9 ± 0.1

310 K

5.0 ± 0.2

3.9 ± 0.1

3.3± 0.1

Importantly, the Arrhenius plot for the lateral diffusion coefficient (Figure 4 inset) shows that, unlike pure DMPC and DMPC with 0.5 mol % alamethicin, DMPC with 0.5 mol % melittin does not exhibit signs of a phase transitions in its dynamic behavior. The uninterrupted Arrhenius temperature dependence of the Dlat demonstrates that the lateral diffusion in DMPC with 0.5 mol % melittin represents the same process at 280 K, 293 K, and 310 K. This is not the case for the other two samples, where the main phase transition remains present, as evidenced by the change in the slope in the Arrhenius plot. For DMPC with melittin, activation energy has been calculated and found to be 5.8 Kcal/mol. For pure DMPC in the fluid phase, activation energy of 7.4 Kcal/mol was observed using pulsed field gradient NMR technique.49 However, these values of activation energy should be compared with care, which always needs to be exercised while comparing the values obtained on different time scales of observation.50 As time scale of observation decreases, molecules experience less of barriers, resulting in lower measured activation energy.50 Lateral diffusion of lipids in membrane depends on various factors, such as free volume, obstructions, interaction between additive and lipids, etc.51-53 The lateral diffusion can be written as D (c ) = D0 f fv (c ) f obst (c ) f int (c ) . Here c is the concentration of additive, D0 is the diffusion coefficient for pure lipid bilayer, factors ffv, fobst, fint, account for a change in the free volume near the additive, direct effect of obstruction and interaction between additive and lipids, respectively. In the fluid phase, it has been shown40 that due to addition of 0.5 mol % peptide, the free volume of phosphocholine membrane remains unaltered; therefore, obstruction effect and interaction between


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peptide and lipids have the major influence on the observed diffusion coefficient. Due to both of these factors, diffusion coefficient decreases, and hence can explain the observed QENS result that in the fluid phase addition of both peptides leads to stiffening of the membrane. Melittin has higher surface charge (+5e) and larger cross section compared to alamethicin (-1e); therefore, a stronger lipid-peptide interaction and obstruction effect is expected for melittin. This could be the reason for the observation that restriction caused by melittin on the lateral diffusion of lipids in the fluid phase is more significant compared to that caused by alamethicin. However, in the ordered phase, addition of 0.5 mol % melittin changes significantly the free volume of Phosphocholine membrane40, which is found to increase. This has been described in terms of local domains of phospholipid, like those in the fluid phase, near the melittin surrounded by the gel phase. Results from small and wide angle X-ray scattering54 also indicate that in the ordered phase, addition of low amount of melittin substantially reduces the long range stacking order of the bilayer. Therefore, in the ordered phase, the factor corresponding to the free volume ffv dominates, which leads to increase in the diffusivity in the case of melittin. However, no such increase in the specific volume of phosphocholine membrane in the gel phase due to addition of alamethicin at 0.5 mol% has been reported. To summarize, we have used elastic and quasielastic incoherent neutron scattering to investigate the effect of antimicrobial peptides on the microscopic dynamics of a phospholipid membrane. We investigated two archetypical AMPs, alamethicin and melittin, at a peptide-to-lipid ratio of 1:200, which was deliberately chosen to be too low to induce formation of stable transmembrane pores. Even at such a low concentration, AMPs demonstrated a profound effect on the lipid dynamics and phase behavior. In particular, melittin, which is known for the relatively stronger interaction with lipids, affected the membrane dynamics to the extent that no signs of the main phase transitions in its dynamic behaviour between the ordered and fluid phases could be observed. On the other hand, alamethicin, which does not interact with the lipids as strongly as melittin, affected the fluid phase transition to a minimal extent, but virtually eliminated the weaker gel-ripple phase transition. Thus, even at the low peptide-to-lipid ratios, insufficient to induce stable transmembrane pores, the microscopic membrane dynamics and membrane phase behavior could be qualitatively altered by AMPs if the AMP-lipid interaction is sufficiently strong.


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Since many important properties of membranes, such as permeability at a given temperature, depend on their phase state, the present study reveals the mechanism by which APMs could act on the pathogens’ membranes even at relatively low concentrations. This can be important from the practical standpoint of AMPs application, where the high AMP concentrations necessary for the transmembrane pore formation may be difficult to attain.

Materials and Methods Alamethicin and Melittin were purchased from Sigma Aldrich (St. Louis, MO). The phospholipid, DMPC was acquired from Avanti Polar Lipids (Alabaster, AL) in the powder form. D2O (99.9%) was obtained from Cambridge Isotope Laboratories (Andover, MA). Unilamellar vesicles solutions of 0.1 M DMPC (a) neat with (b) 0.5 mol % alamethicin and (c) 0.5 mol % melittin were prepared followed the protocol described here55. Elastic incoherent neutron scattering (EINS) and Quasielastic neutron scattering experiments (QENS) have been carried out on all three aforementioned vesicles solution (with and without alamethicin/melittin) using the HFBS45 and BASIS47 spectrometer respectively. Details of EINS and QENS experiments are described here.55 Neutron in-plane scattering experiment was performed on multi-lamellar DMPC, neat and with 0.5 and 4 mol % melittin, using Bio-SANS56.Oriented circular dichroism (OCD) measurements57 were also carried out with the same samples as used for the neutron in-plane experiments on a Jasco (Tokyo, Japan) J-810 spectropolarimeter. Small angle neutron scattering (SANS) measurements were carried out on DMPC ULV with and without 0.5 mol % melittin using Bio-SANS. Details of preparation of sample, in-plane scattering, SANS and OCD experiments are described here.55

Supporting Information Experimental details, detail of analysis of SANS and in-plane scattering data, obtained EISF and HWHM correspond to internal motion are supplied. This information is available free of charge via the Internet at http://pubs.acs.org.


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Note This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC0500OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally

sponsored

research

in

accordance

with

the

DOE

Public

Access

Plan

(http://energy.gov/downloads/doe-public-access-plan). Certain commercial material suppliers are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. The authors declare no competing financial interests. Acknowledgment The neutron scattering experiments on HFBS at NCNR were supported in part by the National Science Foundation under Agreement No. DMR-1508249. The neutron scattering experiments on BASIS at the Spallation Neutron Source were supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The Bio-SANS of the Center for Structural Molecular Biology (FWP ERKP291) at the High Flux Isotope Reactoris supported by the Office of Biological and Environmental Research of the US Department of Energy. ORNL is managed by UT-Battelle, LLC, for the US Department of Energy (DOE) under contract no. DE-AC05- 00OR22725.


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Dynamical and Phase Behavior of a Phospholipid Membrane Altered

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