Effect of an Antimicrobial Peptide on Lateral Segregation of Lipids: A

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Biological and Environmental Phenomena at the Interface

Effect of an Antimicrobial Peptide on Lateral Segregation of Lipids, a Structure and Dynamics Study by Neutron Scattering Veerendra Kumar Sharma, and Shuo Qian Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b04158 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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Effect of an Antimicrobial Peptide on Lateral Segregation of Lipids, a Structure and Dynamics Study by Neutron Scattering Veerendra K. Sharma1**, Shuo Qian2*

Author Affiliation

1Solid

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

2Neutron

Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830

Corresponding Authors *Shuo Qian: [email protected], +1-865-241-1934 **Veerendra K Sharma: [email protected]; +91-22-25594604 Notice (remove before publication): This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC0500OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide

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ABSTRACT Antimicrobial peptides are one of the most promising classes of antibiotic agents for drugresistant bacteria. Although the mechanisms of their action are not fully understood, many of them are found to interact with the target bacterial membrane, causing different degrees of perturbations. In this work, we directly observed a short peptide disturbs membranes by inducing lateral segregation of lipids without forming pores or destroying membranes. Aurein 1.2 (aurein) is a 13-amino acid antimicrobial peptide discovered in the frog Litoria genus that exhibits high antibiotic efficacy. Being cationic and amphiphilic, it binds spontaneously to a membrane surface with or without charged lipids. With a smallangle neutron scattering contrast matching technique that is sensitive to lateral


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heterogeneity in membrane, we found aurein induces significant lateral segregation in an initially uniform lipid bilayer composed of zwitterionic lipid and anionic lipid. More intriguingly, the lateral segregation was similar to the domain formed below the orderdisorder phase transition temperature. To our knowledge, this is the first direct observation of lateral segregation caused by a peptide. With quasielastic neutron scattering, we indeed found that the lipid lateral motion in the fluid phase was reduced even at low aurein concentrations. The reduced lateral mobility makes the membrane prone to additional stresses and defects that change membrane properties and impede membrane-related biological processes. Our results provide insights on how a short peptide kills bacteria at low concentrations without forming pores or destroying membranes. With a better understanding of the interaction, more effective and economically antimicrobial peptides may be designed.


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INTRODUCTION

Antibiotic resistance has become one of the biggest threats to human health, as we are on the brink of losing the battle against ever-increasing drug-resistance microbials.1 Antimicrobial peptides (AMPs), as part of the innate immune system in many species, have shown effectiveness against a broad spectrum of pathogens, including both gram-positive and gram-negative bacteria. Many AMPs have been identified so far from different living organisms.2 Many of them are effective antibiotics that can overcome bacterial resistance; therefore, great effort has been exerted to identify biogenesis AMPs from different species, as well as to engineer AMPs of improved efficacy by applying the motifs learned from the natural peptides. Aurein 1.2 peptide (aurein) is a small AMP with only 13 amino acids that was discovered in the Australian frog Litoria genus.3,4 This short peptide is a very potent AMP with broad-spectrum antibiotic and anticancer activities.3 More intriguingly, it has been found to be an analog to a sequence of human AMP LL-37, LLAA peptide.5,6 The similarity between aurein and LLAA peptide in connection with full length LL-37 makes aurein extremely useful as a model system for gaining general


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knowledge of effective bacteria-killing AMPs. The understanding of such short, yet potent peptides that reproduce antimicrobial functions of much longer-length AMPs will help greatly in designing new and more effective AMPs. Moreover, short peptides are less expensive and easier to produce and dispense; therefore, the insights gained from aurein will aid peptide design suitable for real-world applications.

Although the exact mechanism of AMPs is still under investigation, the membrane is known to be closely associated with AMP function. First, the membrane is an extraordinary barrier for AMPs to overcome to reach any intracellular target to inhibit key biological processes.7 Another prospect, which has been studied extensively, is that the membrane itself is a target of AMPs.8,9 As the enclosures and boundaries of cells and organelles, membranes consist of semipermeable bilayers formed by different lipids, integral/peripheral proteins performing essential functions, and other important molecules such as carbohydrates. The membrane is thought to exist in very fluid phases, decorated by lateral heterogeneity with functional domains that embed certain proteins and lipids.10,11 Disturbances of membranes caused by AMPs, in many ways, hinder normal


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biological functions hence cause trouble for cells. Even worse, some AMPs can compromise the integrity of a membrane by forming membrane pores 12 or disintegrating the lipid bilayers, as in the carpet model.13,14 A major difference between bacterial membranes and mammalian membranes is the composition: first, bacterial membranes do not contain cholesterol, but mammalian membranes do; second, all bacterial membranes contain a significant portion of anionic lipids, whereas mammalian membranes are made of mostly zwitterionic lipids.15–17 The majority of AMPs carry positive charges, which enable a preferential interaction with anionic lipids in membranes as a selectivity mechanism.15 The interaction may cause a detrimental rearrangement of anionic lipids.

18

Aurein is also a cationic AMP. In previous studies, aurein was found to

bind and interact with membranes composed of anionic or zwitterionic lipids, but to have a higher affinity with and be more disruptive on bilayers containing anionic lipids.19–21 Aurein can form a loose bundle as an α-helix over a concentration threshold in the lipid membrane and form peptide/lipid micelles detaching from the membrane.20 Other studies suggest that aurein interacts with lipid membranes primarily on the surface and disrupts the membrane via the carpet model mechanism.

22–24

Our previous study shows that


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aurein doesn’t form membrane pores in bilayers, unlike many other AMPs studied, such as alamethicin and melittin.25 But it causes significant uneven distribution of anionic and zwitterionic lipids between the bilayer leaflets, similar to alamethicin and melittin.26,27 With uneven distribution of lipids, can aurein also promote lateral segregation of anionic lipids and zwitterionic lipids? In this study, we took advantage of a contrast matching technique in small-angle neutron scattering (SANS) to detect the lateral heterogeneity caused by aurein in anionic/zwitterionic lipid compositions.28,29 A lipid bilayer composed of uniformly mixed protiated anionic lipids and deuterated zwitterionic lipids was carefully contrastmatched using certain ratios of a D2O/H2O solution. Any deviation from a laterally uniform mixture would create contrasting regions that could be captured by SANS. Our results show that aurein induces lateral segregation of lipids in the fluid phase, similar to the heterogeneity observed at the lipid gel phase, as if aurein induces a phase change. To our knowledge, this is the first direct observation of such lateral segregation induced by an antimicrobial peptide in membranes. This indicates a significant interaction between the peptide and lipid from the perspectives of lipid molecular dynamics. To further understand it, we used quasielastic neutron scattering (QENS) to investigate the effect of


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aurein on the dynamics of lipid bilayers. We found that, in the fluid phase, the lateral motion of the lipid within the leaflet becomes significantly restricted even with relatively low concentrations of aurein. Our study provides new molecular-level insights into an AMP’s interaction with lipid membrane. Aurein affords us an opportunity to learn from nature how to design effective antibiotic agents that can be used at low concentrations, not to disintegrate membranes but to change the membrane lipid dynamics and promote membrane heterogeneity.

MATERIALS AND METHODS

Materials and Sample Preparation Aurein peptide (>95% purity) was synthesized by GenScript USA Inc. (Piscataway, NJ, USA).

1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine

(d54-DMPC),

1,2-dimyristoyl-sn-

glycero-3-phosphocholine (DMPC), and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, U.S.A.). All materials were used as delivered. Unilamellar vesicles (ULVs) were prepared using the extrusion method as described previously.

26,30

Briefly, required amount of lipid in appropriate molar ratio was taken

in a glass vial and co-dissolved in chloroform, which results a transparent solution. Chloroform was evaporated using the gentle stream of nitrogen gas to obtain lipid films. To remove the residual


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organic solvent, the lipid films were dried by placing the samples under vacuum for about 10 hours at room temperature. Dry lipid films were suspended in the desired amount of D2O/H2O at 320K and went through at least 3 freeze-thaw cycles by alternately placing the lipid suspension in a warm water bath (330K) and in a freezer (253K). ULVs were prepared by extrusion method in which lipid suspension went through a mini-extruder from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) with a porous polycarbonate membrane (pore diameter ~100 nm) more than 20 times. During the extrusion process, the temperature of extruder was kept at ∼320 K to ensure that lipids were in the fluid phase. The stock solution of aurein 10mg/ml was prepared by dissolving it in appropriate D2O/H2O. For SANS experiment, the ULVs solution were made in 2% (w/w) concentration. All SANS samples were stored at 283K before use and were used within 72 hours after preparation. Aurein stock solution was titrated into the vesicle solution to achieve 10 mol%. For QENS, which require more sample amount, the final samples of 7% (w/w) DMPC ULVs solution were prepared by titrating the 100mg/ml lipid stock ULV solution with appropriate amount of aurein stock solution and D2O, to obtain DMPC with aurein in different molar ratios. This mixture was kept at 310K for about 4 hours for equilibration and then used for experiments. Small-Angle Neutron Scattering experiment SANS measurements were performed at the Bio-SANS instrument of Oak Ridge National Laboratory.31 For the contrast variation series and bilayer structure study, the BioSANS is set to 6 Å and a wavelength spread Δλ/λ ~ 0.15 with an effective q-range of 0.003 to 0.8 Å−1. For the domain measurement, the instrument was configured to 18 Å (Δλ/λ ~ 0.15) to provide an effective q-range of ~0.001-0.2 Å−1, which can cover the large structure of the interest. The SANS data from the position sensitive 2D detector was reduced to 1-D profiles I(q) vs. q, by using facility supplied data reduction software Mantid that corrects for detector


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sensitivity, instrument dark current, sample transmission and solvent background. 32 A further constant representing the incoherent background was subtracted from the curves. The temperature was controlled remotely by a water circulation bath with a temperature probe at the sample position. QENS experiment QENS measurements were carried out using high energy resolution backscattering spectrometer BASIS at the Spallation Neutron Source (SNS) of ORNL. 33 For the selected experimental setup, the spectrometer has an energy resolution of 3.4 eV (full width at halfmaximum for the Q-averaged resolution value) and a range of accessible energy transfers of  100 eV suitable for data analysis. The available Q range in the chosen setup was 0.3 Å-1 to 1.9 Å-1. For QENS experiments, the vesicles samples (~2.5 ml) were placed in an annular aluminum sample holder with 0.5 mm internal spacing. These sample holders were selected to give no more than 10% scattering from the samples, thereby minimizing multiple scattering effects. QENS measurements have also been carried out on pure D2O at the same temperatures as a reference sample. In addition, to obtain the spectrometer resolution data, QENS measurements were performed on standard vanadium. MANTID software was used to carry out standard data reduction, which included background subtraction and detector efficiency corrections. 32 QENS data analysis QENS spectra corresponding to the DMPC bilayer are obtained by subtracting the contribution of solvent (D2O) from the ULV solution. The scattered intensity from the lipid membrane, Imem (Q,), can be obtained as, 𝐼𝑚𝑒𝑚(𝑄,𝜔) = 𝐼𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛(𝑄,𝜔) ― 𝜙𝐼𝐷2𝑂(𝑄,𝜔)

(1)


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where  is the volume fraction of D2O. High energy resolution the BASIS spectrometer (E~ 3.4 eV) of the Spallation Neutron Source (SNS) is a well-suited spectrometer to study lateral (~ns) and internal motions (~ps) of the lipid ,

34–37

which is of the current interest. Assuming both

motions are independent of each other, scattering law for the membrane can be written as (2)

𝑆𝑚𝑒𝑚(𝑄,𝜔) = 𝑆𝑙𝑎𝑡(𝑄,𝜔) ⊗ 𝑆𝑖𝑛𝑡(𝑄,𝜔)

where Slat (Q,) and Sint (Q,) are the scattering functions corresponding to lateral and internal motions of the lipid molecules, respectively. The lateral motion of lipids is expected to be characterized by continuous diffusion

34,38.

In that case, scattering law corresponding to lateral

motion, Slat (Q,), would be a Lorentzian function,𝐿𝑙𝑎𝑡(𝛤𝑙𝑎𝑡,𝜔), with Γlat as the half width at halfmaximum (HWHM). The internal motion of lipids is instead localized in nature.34–36 This gives rise to the elastic contribution in the scattering law. Thus, the scattering law for internal motions can be written as 39

(3)

𝑆𝑖𝑛𝑡(𝑄,𝜔) = 𝐴(𝑄)𝛿(𝜔) + (1 ― 𝐴(𝑄))𝐿𝑖𝑛𝑡(𝛤𝑖𝑛𝑡,𝜔)

where the first term represents the elastic part and the second term represents the quasielastic component, which is approximated by a single Lorentzian function. Here A(Q) is Elastic Incoherent Structure Factor (EISF) and Γint is the HWHM of the Lorentzian corresponding to internal motion. Hence, the total scattering law for the membrane can be written as 𝑆𝑚𝑒𝑚(𝑄,𝜔) = [𝐴(𝑄)𝐿𝑙𝑎𝑡(𝛤𝑙𝑎𝑡,𝜔) + (1 ― 𝐴(𝑄))𝐿𝑡𝑜𝑡(𝛤𝑙𝑎𝑡 + 𝛤𝑖𝑛𝑡, 𝜔)]

(4)

For data fitting, Eq. (4) was convoluted with the instrumental resolution function as measured with a vanadium standard, and A(Q), lat, and int were determined by the least squares fitting of the measured spectra. DAVE software developed at the NIST Center for Neutron Research was used for QENS data analysis.40


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RESULTS AND DISCUSSION

Aurein promotes lateral segregation of lipids

In our previous study of aurein on liposomes containing anionic lipids, we found that aurein modified the lipid distribution significantly by drawing an anionic lipid, 1,2dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), to the outer leaflet of the bilayer. At relatively high aurein concentrations, we have found additional feature that cannot be explained by the transversal bilayer structure but was indicative of lateral organization.25 Therefore, we applied the contrast matching SANS in this study to examine it. To take advantage of the neutron scattering contrast between deuterium and hydrogen, we used ULVs made of chain per-deuterated 1,2-dimyristoyl-d-54-sn-glycero-3-phosphocholine (d54-DMPC) and protiated anionic lipid DMPG (3/1 and 1/1 in molar ratio). The contrast in the sample is tunable by titrating the solution with different D2O ratios. Assuming the two lipids were homogenous in lipid bilayers, in a solution of an appropriate D2O ratio such that the neutron scattering length density (NSLD) of the solution matched the


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average NSLD of the lipid bilayer, there would be no coherent scattering intensity except an incoherent flat signal. However, if the lateral homogeneity were disturbed, with the segregation of two lipids in the plane of the lipid bilayer, the contrast between the deuterated and the protiated domains would raise a coherent non-flat scattering intensity. This technique has been successful in detecting nanodomains in model membranes28 and live cell membranes.29

The lipid bilayer contrast matching points of d54-DMPC/DMPG=3/1 and 1/1 were determined to be 66% D2O and 50% D2O, respectively, based on contrast variation measurements at different D2O ratios (see the SI for details). With the determined D2O ratios, we were able to annihilate the characteristic bilayer scattering signal for each lipid composition. As shown by the blue dots in Fig. 1(a, b), the scattering intensity from the lipid-only samples at 313K, above the gel-fluid phase transition temperature of the lipids (~297K), shows an essentially flat signal. It indicates a homogenous mix of d54-DMPC and DMPG in the plane of the membrane in both d54-DMPC/DMPG=3/1 and 1/1 compositions. To confirm the integrity of the lipid bilayer in ULVs, we performed small-


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angle x-ray scattering (SAXS) with the same samples (Fig. S2 in the SI), as the electron density variations between the bilayer and solution could still be probed by X-ray. The SAXS data showed excellent scattering curves from the ULV lipid bilayer structures. The SAXS curves from both lipid compositions were almost identical, as the electron densities from H/D isotopes were the same.

At the same temperature in the fluid phase, the addition of aurein (10 mol%) caused the signal below scattering vector Q0.004 Å-1 to exceed the flat lipid-only scattering signal (313 K, shown as red dots in Fig.1a, b). The significant deviations in scattering intensity can be seen more clearly if the signal for the lipid-only scattering at 313K is subtracted from the signal of the lipid-with-aurein sample for both compositions (Fig. 1c, d). The increase in the scattering intensity originated from the lateral heterogeneity as d54-DMPC and DMPG de-mixed under the influence of aurein. In other words, the anionic lipid was separated from the zwitterionic lipid to form clusters. Aurein promoted the lateral domain in the lipid membrane containing anionic lipid. Based on the Q range of the signal, the size of the lateral domain was estimated to be larger than 70nm


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(~π/0.004 Å-1). We could not tell whether the lateral domain was symmetric across the bilayer leaflets. But based on our previous results on the asymmetric distribution of lipids, it is likely that the domains differed in size between the leaflets. The SAXS data (Fig. S2 in the SI) showed the bilayer with aurein added was still intact, and the addition of aurein shifted the peaks of the curves to the right, indicating a thinning of the lipid bilayer that is consistent with other AMPs.26,41,42 From the peak positions, the membrane thickness changed from 45.86Å-1 (Q=0.137Å-1) in the lipid-only samples to 43.6 Å (Q=0.14 Å-1) with aurein added.

More interestingly, the lipid bilayer with aurein was similar to that without aurein at 283 K, where the lipid was in a gel phase (Fig. 1(a, b), black dots). The deviations from the flat scattering are shown in Fig. 1 (c, d). It appears that aurein induces clustering of anionic lipids, effectively reducing the lipid rearrangement mobility into a gel-like phase even above the main phase transition temperature of the lipids. A functional membrane should be in the fluid phase overall, as lipid molecules are constant reshuffling with molecules such as proteins for different functionalities. The fluidity also reduces the


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tension caused by different molecules to maintain a “defect-free” working mode. If the binding of AMPs, such as aurein, curbs fluidity to promote more defects, the membrane may become vulnerable.18,43 To understand the origin of the limited mobility imposed by aurein in lipids, we studied the dynamics of lipids with and without aurein using the QENS technique.

Figure 1. The SANS scattering intensity from (a) d54-DMPC/DMPG=3/1 without (283 K, 313 K) and with aurein (10 mol %, 313K) in its contrast matching solution with 66%


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D2O; (b) d54-DMPC/DMPG=1/1 without (283K, 313K) and with aurein (10 mol%, 313K) in its contrast matching solution with 50% D2O. The difference in intensity (∆I) was obtained by subtracting the lipid-only sample at 313K (almost flat and close to zero at the contrast matching conditions, as shown in (a, b)) from the respective data set (c) d54-DMPC/DMPG=3/1 samples; (d) d54-DMPC/DMPG=1/1 samples.

Aurein significantly affects the dynamics of lipid bilayers

We systematically studied the effects of aurein on the dynamics of DMPC membranes at different temperatures with QENS to understand how aurein changes the phase behavior of lipids. QENS is one of the most suitable techniques for studying the microscopic dynamics of lipid membranes on picosecond-to-nanosecond time scales and on length scales from angstroms to a few nanometers34,37,44. The DMPC membrane showed a rather weak transition from the gel to the ripple phase at 287K and a strong main (ripple to fluid) phase transition at ~297K.37,45,46 QENS experiments were carried out on DMPC ULV at four distinct temperatures, two below and two above the main phase transition temperature: 280K (gel phase),


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293K (ripple phase), 310K and 320K (fluid phase). To investigate the effects of aurein peptide on the dynamics of the membrane, QENS experiments were also carried out on a DMPC membrane with 1.7 mol% aurein at those temperatures and with two additional concentrations of aurein (1 mol% and 2.5 mol%) at 320K. To verify the binding of the peptide to the lipid bilayer, we measured the circular dichroism (CD) spectra of aurein in DMPC ULVs at the same concentrations and temperatures as in the QENS experiment. The CD spectra show aurein is α-helical with little change below and above the main phase transition temperature (297 K) within the experimental uncertainties (Fig. S4 in the SI). This finding indicates that aurein significantly binds a lipid bilayer composed of only zwitterionic lipids.47 This is consistent with the finding in a previous study that aurein, as a well-defined amphipathic peptide, was able to partition a lipid bilayer well without the presence of charged lipids.47,48 Additionally, SANS experiments were performed to check the DMPC bilayer structure under the influence of aurein. The SANS results showed that aurein thinned the DMPC bilayer in a concentration-dependent manner (see the SI for details). For example, in the gel phase (280K), the total membrane thickness was reduced from 56.3 ±0.6Å to 52.5 ±0.6Å once 1 mol% of aurein was added. In the fluid phase (313K), the membrane thinning effect was lesser: the thickness dropped from 48.6 ±0.6Å to 45.2±0.5Å with 2.5 mol % of aurein added. At the concentrations of aurein used (up to 2.5 mol %), the lipid bilayer was intact as the bilayer structure shown by SANS. QENS spectra corresponding to the DMPC bilayer were obtained by subtracting the contribution of the solvent (D2O) from that of the ULV solution. The resultant subtracted spectra


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for the DMPC bilayer with and without 1.7 mol % aurein are shown in Fig. 2 at 280 K, 293 K, 310 K, and 320 K. Instrument resolution as measured from a standard vanadium sample is also shown in Fig 2. For direct comparison, spectra are normalized to peak amplitude. Significant quasielastic (QE) broadening is observed for the DMPC bilayer with and without aurein, indicating the presence of stochastic motion of the lipid molecules. It is clear from Fig. 2 that there is a sudden large jump in the QE broadening as the temperature rises from 293 K to 310 K, which is due to the main phase transition temperature (297 K) of DMPC. It is evident that adding aurein into the DMPC bilayer leads to a decrease in the QE broadening, especially in the fluid phase of the membrane (310 K and 320 K). This indicates that incorporation of 1.7 mol % aurein affects the microscopic dynamics of the DMPC bilayer. The dynamics of the lipid molecules within the bilayer involve a combination of various motions such as lateral, flip-flop, vibration, conformation, reorientation of individual lipids, and bending or thickness fluctuations of the whole bilayer, encompassing a broad range of time and length scales.44,45,49–52 The QENS spectra with the used configuration (E~ 3.4 eV and available energy transfer  100 eV) can be successfully described with a model in which lipid molecules undergo two distinct motions: (1) lateral motion of the lipid molecule within the leaflet and (2) localized internal motion of the lipid as described in Eq. (4). 34,37


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Figure 2 Peak-normalized QENS spectra at Q =1.1 Å-1 for the neat DMPC bilayer (filled symbols) and DMPC bilayer with 1.7 mol % aurein (open symbols) at 280 K (black pentagon), 293 K (red triangle), 310 K (blue square), and 320 K (green circle). QENS spectra observed from a vanadium standard as a resolution is also shown by a dashed line.

It was found that the above scattering law described the QENS spectra quite well for DMPC bilayers with and without aurein at all the measured temperatures and Q values. Typical fitted spectra for the DMPC bilayer with and without 2.5 mol % aurein at 320K at Q=1.1 Å-1 are shown in Fig. 3 (a) and (b). To gain more insight into the two dynamical processes, the parameters obtained from the fit were analyzed as a function of Q.


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Figure 3 Typical fitted QENS spectra at 320K at Q=1.1 Å-1 for the (a) neat DMPC bilayer and (b) DMPC bilayer with 2.5 mol % aurein.

Lateral motion of lipids was severely restricted: Lateral motion of lipids has paramount importance in many physiological processes, such as cell signaling, permeability, membrane trafficking, membrane fusion, and others. Variations of the HWHM corresponding to lateral motion of the lipid, lat, are shown in Fig. 4. It is evident that at all the temperatures, lat increases linearly with Q2, indicating continuous diffusion described by a simple Brownian motion using Fick’s law, lat = DlatQ2, as shown by lines in Fig. 4. Here, Dlat, the diffusion coefficient for lateral


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motion of DMPC lipid molecules, can be obtained from the slope of the linear fit and is shown in Fig. 5. It was found that aurein significantly affected the lateral motion of the lipid particularly in the fluid phase, which is physiologically relevant. Results are found to be consistent with the fluorescence correlation spectroscopy study which indicated that lateral motion of lipid is restricted due to insertion of antimicrobial peptides.53 In ordered phases (gel and ripple), aurein did not significantly affect the lateral motion of the lipid, as it was already restricted. For example, at 293 K, Dlat for DMPC was found to be 1.60.110-7 cm2/s, which remained almost the same with the inclusion of 1.7 mol % peptide. However, in the fluid phase, the addition of aurein restricted the lateral motion of the lipid significantly. At 310 K, Dlat for lipid was reduced from 6.00.210-7 cm2/s to 4.70.210-7 cm2/s by the addition of 1.7 mol % aurein. The variation of Dlat with the concentration of aurein at 320 K is shown in the inset of Fig. 5. It shows that the lateral diffusion coefficient of lipids decreases monotonously with the increasing concentration of aurein. At 320 K, Dlat for the DMPC membrane is 8.70.310-7 cm2/s, which decreases to 5.40.210-7 cm2/s with the addition of 2.5 mol % aurein.


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Figure 4 Variation of HWHMs corresponding to lateral motion of lipids for (a) the DMPC bilayer with and without 1.7 mol % aurein at 280 K, 293 K, and 310 K. (b) The DMPC bilayer with different concentrations of aurein at 320 K.


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Figure 5 Lateral diffusion coefficient, Dlat, for the DMPC bilayer with and without 1.7 mol % aurein at different temperatures. It is evident that the stiffening effect of aurein is more prominent in the fluid phase (310, 320 K). The variation of Dlat with different concentrations of aurein at 320 K is shown in the inset.

Internal motion of lipids was not affected: The internal motion of lipids is complex and comprises various motions, such as rotational, conformational change, torsional motion, and others. EISF, A(Q) and HWHM of the Lorentzian, int, corresponding to the internal motion of the DMPC with and without 1.7 mol % aurein at 280K, 293K, 310K and 320 K are shown in Fig. 6. A(Q) and int for the DMPC only and with two peptide concentrations, 1 mol % and 2.5 mol %, at 320K are shown in the inset. The internal motion of lipids at 280 K (gel phase) and at all the other temperatures (293 K, 310 K, and 320 K) was described very well using the uniaxial rotational diffusion (URD) and localized translational diffusion (LTD) models, respectively. Details for the URD and LTD models are discussed in the SI. In URD model, it is assumed that a fraction of hydrogen atoms of the lipids undergoes uniaxial rotation around the molecular axes. In this case, 1

𝑁

resultant EISF can be written as 𝑝𝑥𝑁∑𝑝 = 1𝑗0(2𝑄𝑟𝑠𝑖𝑛

𝜋𝑝 𝑁)

+(1 ― 𝑝𝑥) where px is the fraction of

mobile hydrogen atoms and r is the radius of circle on which hydrogen atoms rotates. It is found that URD model describes the data well as shown by lines in Fig. 6 and the fitting parameters are listed in Table 1. The results show that in gel phase, 53% of hydrogens in the lipid molecule


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undergo a uniaxial rotation on a circle of radius 1.7 Å with a rotational diffusion coefficient of 11.4 μeV. The addition of aurein does not change the parameters significantly; indicating, it has a little impact on internal motion of DMPC in gel phase.

LTD model has been used to describe the dynamics of DMPC bilayer at 293 and 310K with and without aurein. In this model, hydrogen atoms undergo localized translational diffusion within spheres of different sizes and associated diffusivities. We have assumed a simple linear distribution of the size and diffusivity along the alkyl chain. Hydrogen atoms in first CH2 unit nearest the head group faces maximum constrained hence has smallest radius of sphere, Rmin and diffusivity Dmin. Size and diffusivity increase with the alkyl chain and it would be maximum at the last CH3 unit of the alkyl chain. It is found that LTD model describes the data well as shown by lines in Fig. 6 and the fitting parameters are listed in Table 2. For DMPC only at 320K, the localized motions of the hydrogen atoms along the acyl chains are restricted to spheres of radii varying between 0.3 to 5.2 Å, with corresponding diffusion coefficient ranging from 1.7 to 7810-7 cm2/s. Small values of Rmin and Dmin should be taken with reservation; they merely reflect the negligible movement of the hydrogens in the first carbon position held by the headgroup. It is evident that the addition of aurein do not significantly affect the internal motions of the membrane except at 320 K, where a modest hindrance in the internal motion is observed.


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Figure 6 Variation of (a) EISF and (b) HWHM corresponding to internal motion of lipid in DMPC only (filled symbols) and DMPC with 1.7 mol % aurein (empty symbols) at 280 (square), 293 (circle), 310 (triangle) and 320 K (diamond). EISF and HWHM corresponding to internal motion of DMPC with aurein at 320K at concentrations of 1 mol % (bottom half filled diamond) and 2.5 mol % (top half-filled diamond) are shown in the inset. Lines correspond to the model fits as described in text.

Table 1 Fraction of mobile hydrogen, radius of circle and rotational diffusion coefficient as obtained from the least square fitting of EISF and HWHM, int at 280K, assuming uniaxial rotational diffusion model.

px (fraction of mobile hydrogens)

DMPC only

DMPC+ 1.7 mol % aurein

533 %

522 %


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r (radius of circle)

1.70.1 Å

1.60.1 Å

Dr (rotational diffusion coefficient) 11.40.6 eV

11.7 0.7 eV

Table 2 Maximum confining radius (Rmax) and associated diffusion coefficient (Dmax) as obtained from the least square fitting of EISF and HWHM, int at 293K, 310K and 320K, assuming localized translational diffusion model. T

DMPC only

DMPC+1.7 mol %

DMPC+1 mol %

DMPC+2.5 mol %

aurein

aurein

aurein

(K) Rmax

Dmax

Rmax

Dmax

Rmax

Dmax

Rmax

Dmax

(Å)

(10-7

(Å)

(10-7

(Å)

(10-7

(Å)

(10-7

cm2/s)

cm2/s)

cm2/s)

cm2/s)

293K 2.90.1

251

2.90.1

261

-

-

-

-

310K 5.00.2

562

5.00.2

563

-

-

-

-

320K 5.20.2

783

5.30.2

752

5.20.2

763

5.30.3

722

While aurein interacts more strongly with charged lipids, as shown by our previous study and other studies, 23,25,48 there is still considerable interaction with zwitterionic lipids, a finding sustained by our results from SANS, CD, and QENS on membranes composed of a single zwitterionic lipid. The phenylalanine within aurein favors anchoring to the membrane

6,20,54,

and the hydrophobicity of the peptide drives it to integrate into the


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bilayer. The different phases of DMPC didn’t affect the binding, as the CD data showed essentially no change in aurein’s helicity below and above the main phase transition temperature. Once aurein bound to the lipid bilayer, presumably mostly at the headgroupchain interface region at the concentrations in our QENS experiment,

20,25

it significantly

altered the dynamics of the membrane, especially in the fluid phase. This indicates that aurein, like many other AMPs, binds and modulates the lipid dynamics regardless of lipid charge.12,35,37 While aurein causes the membrane thinning as shown by SAXS and SANS, the effect is limited as aurein doesn’t induce membrane pore.25 Compared to bilayers with charged lipid, the restriction on the lateral motion might not be as antagonistic in zwitterionic lipid bilayers, since there is no charged lipid to interact cooperatively with aurein into further destruction.

There are more implications for membranes with charged lipids. The QENS results showed that the addition of aurein restricted mainly the lipid lateral motion, not the internal motion. In the fluid phase, we saw a concentration dependence of the restriction, i.e., as the concentration of aurein increased, the lateral diffusion slowed gradually. The


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slowdown has a few consequences. First, in a membrane composed of different types of lipids, the homogeneity decreases, with possible de-mixing of charged lipids or coexistence of phases. The preferable interaction of charged lipid and peptide can exacerbate this effect. This could cause the nanoscopic lateral domains we saw in the contrast matching SANS experiment.55 Second, the membrane became more prone to defect even above the main phase transition temperature, as the slower lateral motion restricted the membrane from recovering from any stresses or defects caused by intruding agents, or even normal cell processes. All these changes have adverse effects on bacterial membranes and their membrane proteins that rely on a proper membrane environment to function normally.

CONCLUSIONS

The present study provides new molecular-level insights into the mechanism of an AMP on lipid membrane. This mechanism requires no membrane pore formation or membrane disintegration. With or without charged lipids, aurein readily binds to a lipid membrane spontaneously with well-defined hydrophobic and hydrophilic sides.


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Phenylalanine residues and electrostatic interaction with the anionic lipid both can further promote binding.20 The amphiphilic aurein stays at the headgroup-chain interfacial region, with different orientations at different concentrations. 25 With shorter lengths compared with other AMPs, transmembrane pore formation is hardly possible, as shown in our previous study. 25 But short peptides can assemble into different oligomers, 20 which are adaptable to different sizes to interact with charged lipids within certain ranges laterally on a membrane. 18 Such electrostatic interactions promote a preferable interaction of aurein with an anionic lipid in the form of asymmetric bilayers and lateral segregation of charged and zwitterionic lipids, evident from the direct structural results from contrast-matching SANS. As aurein reduces the lateral diffusion of lipid in the membrane, it hinders the ability of lipids to rearrange to accommodate increased membrane tension and other physical properties required by normal membrane functions. Moreover, the higher concentration of aurein associated with charged lipids settles the charged-lipid rich domain that is different from the surrounding lipid bilayer. This may create a highly stressed phase boundary defect that increases permeability in a way disastrous to the membrane. 18,48,56 Such defects also enable


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aurein to penetrate through membranes. This finding explains why aurein is a good “cargo carrier,” as shown in a previous study. 57

ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge in the Supporting Information on ACS publication website. The SI includes contrast matching measurements, SAXS, CD and analysis methods for QENS data.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS The Spallation Neutron Source, where BASIS is located, and the High Flux Isotope Reactor, where Bio-SANS is located, are supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. The Bio-SANS of the


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Center for Structural Molecular Biology at the High Flux Isotope Reactor is supported by the Office of Biological and Environmental Research of the US Department of Energy. The D2O used in this research was supplied by the U.S. Department of Energy the Isotope Program in the Office of Nuclear Physics. We thank Eugene Mamontov for assistance on the BASIS instrument. REFERENCES (1)

Martens, E.; Demain, A. L. The Antibiotic Resistance Crisis, with a Focus on the United States. The Journal of Antibiotics 2017, 70 (5), 520–526.

(2)

Wang, G.; Li, X.; Wang, Z. APD3: The Antimicrobial Peptide Database as a Tool for Research and Education. Nucleic Acids Res. 2016, 44 (D1), D1087-1093.

(3)

Rozek, T.; Wegener, K. L.; Bowie, J. H.; Olver, I. N.; Carver, J. A.; Wallace, J. C.; Tyler, M. J. The Antibiotic and Anticancer Active Aurein Peptides from the Australian Bell Frogs Litoria Aurea and Litoria Raniformis the Solution Structure of Aurein 1.2. Eur. J. Biochem. 2000, 267 (17), 5330–5341.

(4)

Seto, G. W. J.; Marwaha, S.; Kobewka, D. M.; Lewis, R. N. A. H.; Separovic, F.; McElhaney, R. N. Interactions of the Australian Tree Frog Antimicrobial Peptides Aurein 1.2, Citropin 1.1 and Maculatin 1.1 with Lipid Model Membranes: Differential Scanning Calorimetric and Fourier Transform Infrared Spectroscopic Studies.

Biochimica et Biophysica Acta (BBA) - Biomembranes 2007, 1768 (11), 2787–2800. (5)

Li, X.; Li, Y.; Han, H.; Miller, D. W.; Wang, G. Solution Structures of Human LL-37 Fragments and NMR-Based Identification of a Minimal Membrane-Targeting Antimicrobial and Anticancer Region. J. Am. Chem. Soc. 2006, 128 (17), 5776– 5785.

(6)

Li, X.; Li, Y.; Peterkofsky, A.; Wang, G. NMR Studies of Aurein 1.2 Analogs.

Biochimica et Biophysica Acta (BBA) - Biomembranes 2006, 1758 (9), 1203–1214. (7)

Le, C.-F.; Fang, C.-M.; Sekaran, S. D. Intracellular Targeting Mechanisms by Antimicrobial Peptides. Antimicrob. Agents Chemother. 2017, 61 (4).

(8)

Zasloff, M. Antimicrobial Peptides of Multicellular Organisms. Nature 2002, 415 (6870), 389–395.


32

Page 33 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(9)

Melo, M. N.; Ferre, R.; Castanho, M. A. R. B. Antimicrobial Peptides: Linking Partition, Activity and High Membrane-Bound Concentrations. Nature Reviews

Microbiology 2009, 7 (3), 245–250. (10) Singer, S. J.; Nicolson, G. L. The Fluid Mosaic Model of the Structure of Cell Membranes. Science 1972, 175 (4023), 720–731. (11) Goñi, F. M. The Basic Structure and Dynamics of Cell Membranes: An Update of the Singer–Nicolson Model. Biochimica et Biophysica Acta (BBA) - Biomembranes 2014, 1838 (6), 1467–1476. (12) Huang, H. W.; Chen, F.-Y.; Lee, M.-T. Molecular Mechanism of Peptide-Induced Pores in Membranes. Phys. Rev. Lett. 2004, 92 (19), 198304. (13) Shai, Y. Mechanism of the Binding, Insertion and Destabilization of Phospholipid Bilayer Membranes by Alpha-Helical Antimicrobial and Cell Non-Selective Membrane-Lytic Peptides. Biochim. Biophys. Acta 1999, 1462 (1–2), 55–70. (14) Shai, Y. Mode of Action of Membrane Active Antimicrobial Peptides. Peptide

Science 2002, 66 (4), 236–248. (15) Epand, R. F.; Savage, P. B.; Epand, R. M. Bacterial Lipid Composition and the Antimicrobial Efficacy of Cationic Steroid Compounds (Ceragenins). Biochimica et

Biophysica Acta (BBA) - Biomembranes 2007, 1768 (10), 2500–2509. (16) Sohlenkamp, C.; Geiger, O. Bacterial Membrane Lipids: Diversity in Structures and Pathways. FEMS Microbiol Rev 2016, 40 (1), 133–159. (17) Meer, G. van; Kroon, A. I. P. M. de. Lipid Map of the Mammalian Cell. J Cell Sci 2011, 124 (1), 5–8. (18) Epand, R. M.; Epand, R. F. Lipid Domains in Bacterial Membranes and the Action of Antimicrobial Agents. Biochimica et Biophysica Acta (BBA) - Biomembranes 2009, 1788 (1), 289–294. (19) Ambroggio, E. E.; Separovic, F.; Bowie, J. H.; Fidelio, G. D.; Bagatolli, L. A. Direct Visualization of Membrane Leakage Induced by the Antibiotic Peptides: Maculatin, Citropin, and Aurein. Biophysical Journal 2005, 89 (3), 1874–1881. (20) Shahmiri, M.; Enciso, M.; Mechler, A. Controls and Constrains of the Membrane Disrupting Action of Aurein 1.2. Scientific Reports 2015, 5, 16378.


33

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 44

(21) Laadhari, M.; Arnold, A. A.; Gravel, A. E.; Separovic, F.; Marcotte, I. Interaction of the Antimicrobial Peptides Caerin 1.1 and Aurein 1.2 with Intact Bacteria by 2H Solid-State NMR. Biochimica et Biophysica Acta (BBA) - Biomembranes 2016,

1858 (12), 2959–2964. (22) Fernandez, D. I.; Brun, A. P. L.; Whitwell, T. C.; Sani, M.-A.; James, M.; Separovic, F. The Antimicrobial Peptide Aurein 1.2 Disrupts Model Membranes via the Carpet Mechanism. Phys. Chem. Chem. Phys. 2012, 14 (45), 15739–15751. (23) Lee, T.-H.; Heng, C.; Swann, M. J.; Gehman, J. D.; Separovic, F.; Aguilar, M.-I. Real-Time Quantitative Analysis of Lipid Disordering by Aurein 1.2 during Membrane Adsorption, Destabilisation and Lysis. Biochimica et Biophysica Acta

(BBA) - Biomembranes 2010, 1798 (10), 1977–1986. (24) Mechler, A.; Praporski, S.; Atmuri, K.; Boland, M.; Separovic, F.; Martin, L. L. Specific and Selective Peptide-Membrane Interactions Revealed Using Quartz Crystal Microbalance. Biophys. J. 2007, 93 (11), 3907–3916. (25) Rai, D. K.; Qian, S. Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer. Scientific Reports 2017, 7 (1), 3719. (26) Qian, S.; Heller, W. T. Peptide-Induced Asymmetric Distribution of Charged Lipids in a Vesicle Bilayer Revealed by Small-Angle Neutron Scattering. The Journal of

Physical Chemistry B 2011, 115 (32), 9831–9837. (27) Rai, D. K.; Qian, S.; Heller, W. T. The Interaction of Melittin with Dimyristoyl Phosphatidylcholine-Dimyristoyl Phosphatidylserine Lipid Bilayer Membranes.

Biochimica et Biophysica Acta (BBA) - Biomembranes 2016, 1858 (11), 2788–2794. (28) Heberle, F. A.; Petruzielo, R. S.; Pan, J.; Drazba, P.; Kučerka, N.; Standaert, R. F.; Feigenson, G. W.; Katsaras, J. Bilayer Thickness Mismatch Controls Domain Size in Model Membranes. J. Am. Chem. Soc. 2013, 135 (18), 6853–6859. (29) Nickels, J. D.; Chatterjee, S.; Stanley, C. B.; Qian, S.; Cheng, X.; Myles, D. A. A.; Standaert, R. F.; Elkins, J. G.; Katsaras, J. The in Vivo Structure of Biological Membranes and Evidence for Lipid Domains. PLOS Biology 2017, 15 (5), e2002214.


34

Page 35 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(30) Qian, S.; Rai, D.; Heller, W. T. Alamethicin Disrupts the Cholesterol Distribution in Dimyristoyl Phosphatidylcholine–Cholesterol Lipid Bilayers. J. Phys. Chem. B 2014,

118 (38), 11200–11208.. (31) Heller, W. T.; Urban, V. S.; Lynn, G. W.; Weiss, K. L.; O’Neill, H. M.; Pingali, S. V.; Qian, S.; Littrell, K. C.; Melnichenko, Y. B.; Buchanan, M. V.; et al. The Bio-SANS Instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory.

Journal of Applied Crystallography 2014, 47 (4), 1238–1246. (32) Arnold, O.; Bilheux, J. C.; Borreguero, J. M.; Buts, A.; Campbell, S. I.; Chapon, L.; Doucet, M.; Draper, N.; Ferraz Leal, R.; Gigg, M. A.; et al. Mantid—Data Analysis and Visualization Package for Neutron Scattering and SR Experiments. Nuclear

Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 2014, 764, 156–166. (33) Mamontov, E.; Herwig, K. W. A Time-of-Flight Backscattering Spectrometer at the Spallation Neutron Source, BASIS. Review of Scientific Instruments 2011, 82 (8), 085109. (34) Sharma, V. K.; Mamontov, E.; Anunciado, D. B.; O’Neill, H.; Urban, V. Nanoscopic Dynamics of Phospholipid in Unilamellar Vesicles: Effect of Gel to Fluid Phase Transition. J. Phys. Chem. B 2015, 119 (12), 4460–4470. (35) K. Sharma, V.; Mamontov, E.; B. Anunciado, D.; O’Neill, H.; S. Urban, V. Effect of Antimicrobial Peptide on the Dynamics of Phosphocholine Membrane: Role of Cholesterol and Physical State of Bilayer. Soft Matter 2015, 11 (34), 6755–6767. (36) Sharma, V. K.; Mamontov, E.; Tyagi, M.; Urban, V. S. Effect of α-Tocopherol on the Microscopic Dynamics of Dimyristoylphosphatidylcholine Membrane. J. Phys.

Chem. B 2016, 120 (1), 154–163. (37) Sharma, V. K.; Mamontov, E.; Tyagi, M.; Qian, S.; Rai, D. K.; Urban, V. S. Dynamical and Phase Behavior of a Phospholipid Membrane Altered by an Antimicrobial Peptide at Low Concentration. J. Phys. Chem. Lett. 2016, 7 (13), 2394–2401. (38) Armstrong, C. L.; Trapp, M.; Peters, J.; Seydel, T.; Rheinstädter, M. C. Short Range Ballistic Motion in Fluid Lipid Bilayers Studied by Quasi-Elastic Neutron Scattering.

Soft Matter 2011, 7 (18), 8358–8362.


35

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Page 36 of 44

(39) Bee, M. Quasielastic Neutron Scattering. 1988. (40) Azuah, R. T.; Kneller, L. R.; Qiu, Y.; Tregenna-Piggott, P. L. W.; Brown, C. M.; Copley, J. R. D.; Dimeo, R. M. DAVE: A Comprehensive Software Suite for the Reduction, Visualization, and Analysis of Low Energy Neutron Spectroscopic Data.

J Res Natl Inst Stand Technol 2009, 114 (6), 341–358. (41) Huang, H. W. Action of Antimicrobial Peptides: Two-State Model. Biochemistry 2000, 39 (29), 8347–8352. (42) Mecke, A.; Lee, D.-K.; Ramamoorthy, A.; Orr, B. G.; Holl, M. M. B. Membrane Thinning Due to Antimicrobial Peptide Binding: An Atomic Force Microscopy Study of MSI-78 in Lipid Bilayers. Biophysical Journal 2005, 89 (6), 4043–4050. (43) Wadhwani, P.; Epand, R. F.; Heidenreich, N.; Bürck, J.; Ulrich, A. S.; Epand, R. M. Membrane-Active Peptides and the Clustering of Anionic Lipids. Biophysical

Journal 2012, 103 (2), 265–274. (44) Busch, S.; Smuda, C.; Pardo, L. C.; Unruh, T. Molecular Mechanism of Long-Range Diffusion in Phospholipid Membranes Studied by Quasielastic Neutron Scattering.

J. Am. Chem. Soc. 2010, 132 (10), 3232–3233. (45) Sharma, V. K.; Mamontov, E.; Ohl, M.; Tyagi, M. Incorporation of Aspirin Modulates the Dynamical and Phase Behavior of the Phospholipid Membrane. Phys. Chem.

Chem. Phys. 2017, 19 (3), 2514–2524. (46) Heimburg, T. A Model for the Lipid Pretransition: Coupling of Ripple Formation with the Chain-Melting Transition. Biophys. J. 2000, 78 (3), 1154–1165. (47) Lorenzón, E. N.; Riske, K. A.; Troiano, G. F.; Da Hora, G. C. A.; Soares, T. A.; Cilli, E. M. Effect of Dimerization on the Mechanism of Action of Aurein 1.2. Biochimica

et Biophysica Acta (BBA) - Biomembranes 2016, 1858 (6), 1129–1138. (48) Fernandez, D. I.; Sani, M.-A.; Miles, A. J.; Wallace, B. A.; Separovic, F. Membrane Defects Enhance the Interaction of Antimicrobial Peptides, Aurein 1.2 versus Caerin 1.1. Biochimica et Biophysica Acta (BBA) - Biomembranes 2013, 1828 (8), 1863– 1872. (49) Nagao, M.; Kelley, E. G.; Ashkar, R.; Bradbury, R.; Butler, P. D. Probing Elastic and Viscous Properties of Phospholipid Bilayers Using Neutron Spin Echo Spectroscopy. J. Phys. Chem. Lett. 2017, 8 (19), 4679–4684.


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(50) Reinhard Lipowsky; Sackmann E. Handbook of Biological Physics | Structure and

Dynamics of Membranes - From Cells to Vesicles | ScienceDirect.Com; North Holland, 1995. (51) Tocanne, J. F.; Dupou-Cézanne, L.; Lopez, A. Lateral Diffusion of Lipids in Model and Natural Membranes. Prog. Lipid Res. 1994, 33 (3), 203–237. (52) Marquardt, D.; Heberle, F. A.; Miti, T.; Eicher, B.; London, E.; Katsaras, J.; Pabst, G. 1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers. Langmuir 2017, 33 (15), 3731–3741. (53) Macháň, R.; Hof, M. Lipid Diffusion in Planar Membranes Investigated by Fluorescence Correlation Spectroscopy. Biochimica et Biophysica Acta (BBA) -

Biomembranes 2010, 1798 (7), 1377–1391. (54) MacCallum, J. L.; Bennett, W. F. D.; Tieleman, D. P. Distribution of Amino Acids in a Lipid Bilayer from Computer Simulations. Biophysical Journal 2008, 94 (9), 3393– 3404. (55) Tocanne, J.-F.; Dupou-Cézanne, L.; Lopez, A.; Tournier, J.-F. Lipid Lateral Diffusion and Membrane Organization. FEBS Letters 257 (1), 10–16. (56) Cordeiro, R. M. Molecular Structure and Permeability at the Interface between Phase-Separated Membrane Domains. J. Phys. Chem. B 2018, 122 (27), 6954– 6965. (57) Li, M.; Tao, Y.; Shu, Y.; LaRochelle, J. R.; Steinauer, A.; Thompson, D.; Schepartz, A.; Chen, Z.-Y.; Liu, D. R. Discovery and Characterization of a Peptide That Enhances Endosomal Escape of Delivered Proteins in Vitro and in Vivo. J. Am.

Chem. Soc. 2015, 137 (44), 14084–14093.


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Table of Contents (TOC) Graphic

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The interaction of aurein and lipids: (a) aurein causes lateral segregation of anionic lipid in membranes; (b) aurein reduces the lateral diffusion of lipid in membranes


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Figure 1 406x203mm (300 x 300 DPI)


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QENS data Fits Lateral Lateral+Internal Background

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Figure 5 365x272mm (96 x 96 DPI)


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1.0

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Effect of an Antimicrobial Peptide on Lateral Segregation of Lipids: A

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