Gramicidin Peptides Alter Global Lipid Compositions and Bilayer

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Gramicidin Peptides Alter Global Lipid Compositions And Bilayer Thicknesses Of Coexisting Liquid-Ordered And Liquid-Disordered Membrane Domains Ebrahim Hassan-Zadeh, Fazle Hussain, and Juyang Huang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03688 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017

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Gramicidin Peptides Alter Global Lipid Compositions And Bilayer Thicknesses Of Coexisting Liquid-Ordered And Liquid-Disordered Membrane Domains Ebrahim Hassan-Zadeh†,#, Fazle Hussain‡, and Juyang Huang*,† †

Department of Physics, Texas Tech University, Lubbock, Texas 79409, United States Department of Physics, Mahshahr Branch, Islamic Azad University, Mahshahr, Iran ‡ Department of Mechanical Engineering, Texas Tech University, Lubbock, Texas 79409, United States #

*Corresponding Author: Juyang Huang, Department of Physics, Texas Tech University, Lubbock, TX 79409, USA; Tel.: (806) 834-3182; Fax: (806)742-1182; Email: [email protected]

Keywords: Membrane domain, lipid phase diagram, thermodynamic tie-line, cholesterol, partition coefficient

1 ACS Paragon Plus Environment

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ABSTRACT Effects of adding 1 mol% of gramicidin-A on the biochemical properties of coexisting liquid-ordered and liquid-disordered (Lo+Ld) membrane domains were investigated. Quaternary giant unilamellar vesicles (GUV) of di18:1PC(DOPC)/di18:0PC(DSPC)/cholesterol/gramicidin-A were prepared using our recently developed damp-film method. The phase boundary of Lo+Ld coexisting region was determined using video fluorescence microscopy. Through fitting Nile Red fluorescence emission spectra, the thermodynamic tie-lines in the Lo+Ld 2-phase region were determined. We found that at 1 mol% (i.e., ~7% of membrane area), gramicidin peptides alter the phase boundary and thermodynamic tie-lines. Gramicidin abolishes the coexisting phases at some lipid compositions, but induces phase separation at others. Previous studies of gramicidin emphasize the local perturbation of bilayer thickness adjacent to the protein through the interaction of “hydrophobic mismatch”. For the first time, it becomes clear that adding gramicidin produces significant long-range and global effects on the structure of membrane domains: it alters the overall lipid compositions and bilayer thicknesses of coexisting Lo and Ld domains. We also found that gramicidin partitions favorably into the Ld phase. Adding gramicidin decreases cholesterol in the Ld phase and increases cholesterol in the Lo phase. Those compositional changes broaden the bilayer thickness difference between Lo and Ld domains, and facilitate preferential partition of gramicidin into thinner Ld domains. Our results demonstrate that membrane proteins play significant roles in determining lipid compositions and bilayer thicknesses of biomembrane domains.


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INTRODUCTION Membrane Proteins and lipids do not mix uniformly in cell plasma membranes, but form sub-resolution microdomains that differ in molecular composition, fluidity, and bilayer thickness.1 In a recent study of plasma membrane of intact, live cells, Owen et al. concluded that the membrane has approximately 76% ordered and 24% disordered subresolution domains.2 Lipid rafts are hypothesized microdomains in cell membranes involved in regulating many vital cellular processes, such as signal transduction, lipid trafficking and protein function.3-5 In the past two decades, a large number of studies have focused on protein-free model membranes composed of lipids and cholesterol.6-9 It has been demonstrated that even a ternary lipid mixture can have surprisingly complex phase behavior. In model membranes, lipid rafts are often assumed to be the liquidordered (Lo) phase, which is rich in cholesterol and high-melting (gel-phase) lipids with saturated acyl chains - coexisting with the liquid-disordered (Ld) phase, which is rich in low-melting (fluid-phase) lipids with unsaturated acyl chains. Lo domains typically have greater bilayer thicknesses and tighter lipid packing than Ld domains. Biomembranes are crowded with proteins. For human red blood cell (RBC) plasma membrane, proteins cover 23% of membrane area.10 In other membranes, estimations go as high as 50%.10,11 By depleting either cholesterol or proteins from cell plasma membranes, Mitra et al.12 concluded that membrane proteins rather than cholesterol molecules modulate the average bilayer thickness of eukaryotic cell membranes. Protein-lipid interactions have been shown to be important to the topology and function of polytopic lactose permease.13 Compared to pure lipid bilayers, lipid bilayers containing proteins are far less studied, largely due to the difficulties in incorporating proteins into membranes and in performing precise quantitative analyses.14 The best-documented protein-lipid interaction is the “hydrophobic mismatch”, i.e., the hydrophobic thickness of a lipid bilayer needing to match the hydrophobic length of trans-membrane proteins for proper protein functions.15-21 Lipids next to a protein are either compressed or stretched for this matching. Thus, hydrophobic mismatch can


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induce perturbations in local bilayer thickness as well as in protein conformation. It is well documented that bilayer thickness can regulate membrane protein functions.20, 22 Model membranes offer an excellent platform to study effects of membrane components under well-controlled conditions. Although the structure of compositionally simple binary and ternary lipid systems have been extensively studied in the last three decades,6-9 investigations of protein-lipid interactions in compositional complex membrane system containing coexisting Lo+Ld domains are quite rare - both experimentally and computationally. Generally speaking, if a lipid mixture only has a single phase, either Lo or Ld, protein-lipid interactions would not be able to change the protein concentration or overall lipid composition of the membrane. However, if a lipid mixture contains coexisting Lo+Ld membrane domains, it becomes possible for proteins to alter the lipid compositions and relative amounts of both types of domains. Furthermore, a protein may show preferential partition to a particular type of domain. Therefore, a lipid mixture containing coexisting phases is a better model system for studying protein-lipid interactions in biomembranes, which have many microdomains. At present, it is not well understood how proteins-lipid interactions contribute to the sizes, overall bilayer thicknesses, and compositions of biomembrane domains. Here we study the effects of adding 1 mol% of gramicidin-A (gA) peptides to the well-studied di18:1PC(DOPC)/di18:0PC(DSPC)/cholesterol lipid mixtures containing coexisting Ld and Lo domains. Gramicidin-A is a hydrophobic linear pentadecapeptide produced by the Bacillus brevis bacterium. Gramicidin forms transmembrane channels that are specific for monovalent cations and is the simplest and the most studied membrane peptide. By measuring phase boundaries and thermodynamic tie-lines with and without gramicidin, and protein partition coefficients in phase separated liposomes, the effects of gramicidin on membrane domain properties are systematically quantified. To the best of our knowledge, this work is the first study of the effect of gA on Lo+Ld phase boundaries and compositions of membrane domains. We found that at 1 mol%, gramicidin peptides alter the phase boundary and thermodynamic tie-lines. Our data show


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that adding gramicidin produces significant long-range and global effects on membrane domain structure: it alters the overall lipid compositions, and thus the bilayers thicknesses of coexisting Lo and Ld domains. Our gramicidin partition coefficient measurement indicates that gramicidin partitions favorably into the Ld phase. Adding gramicidin decreases cholesterol in the Ld phase, and the effect is opposite in the Lo phase. Those compositional changes broaden the bilayer thickness difference between the two types of domains, and facilitate preferential partition of gramicidin into the thinner Ld domains. Our results demonstrate that membrane proteins play significant roles in determining lipid compositions and bilayer thicknesses of biomembrane domains. MATERIALS AND METHODS Materials

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-

glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Gramicidin-A, purchased from Sigma Chemical Co. (St. Louis, MO), was used without further purification. 1,1'-Didodecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiIC12(3)) was purchased from Invitrogen (Carlsbad, CA). 5HBenzo[α]phenoxazin-5-one (Nile Red) was purchased from Molecular Probes (Eugene, OR). Cholesterol was purchased from Nu Chek Prep, Inc. (Elysian, MN). Purity of the phospholipids (>99%) was confirmed by thin layer chromatography (TLC) on washed, activated silica gel plates (Alltech Associates, Deerfield, IL) and developed with a 65:25:4 chloroform/methanol/water mixture. The molarities of the phospholipid stocks were determined by phosphate assay.23 Indium Tin Oxide (ITO) coated slides were purchased from Delta Technologies (Loveland, Colorado). Preparation of GUVs by Electroformation from Damp Lipid Film Quaternary (DOPC/DSPC/cholesterol/gramicidin-A) giant unilamellar vesicles (GUVs) were prepared using our recently developed damp-film method.24,25 We have shown that GUVs prepared by this method have better compositional uniformity. In this procedure, we avoid dry-lipid film state in order to reduce lipid demixing. We first prepare liposomes using the updated Rapid Solvent Exchange (RSE) method,26 then use RSE


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liposomes to produce damp lipid films, and finally use the electroformation method27 to produce GUVs from the damp lipid films. For fluorescence microscopy experiments, all GUVs were labeled with 0.5 mol% of DiIC12 (3) florescence dye, which preferentially partitions into the liquid-disordered (Ld) lipid domains.24,25 Determining The Phase Boundary of Lo+Ld Coexisting Region Fluorescence images and videos of GUVs were captured using a Cooke SensiCam CCD camera (Auburn Hills, MI) on an Olympus IX70 inverted microscope (Melville, NY) with a 40X Olympus objective. The samples were placed on a home-built sample stage made of copper and aluminum, with the sample in direct contact with the copper part. Samples were heated by two thermoelectric Peltier modules (06311-5L31-02CFL, Custom Thermoelectric, Bishopville, MD) controlled by a thermoelectric temperature controller and a 10 k thermistor (WTC3293-14001-A and TCS10K5, Wavelength Electronics, Bozeman, MT). All images and videos were captured at 25 C. The mole fraction of gA in all samples was fixed at 1% based on the molecular weight of gA. The lipid composition is specified by two parameters: the R value [R= DSPC /(DSPC + DOPC)] and cholesterol mole fraction C. In order to simplify the comparison of results with and without gramicidin, gramicidin is not included in lipid composition calculation. Several measures were taken to avoid the problem of light-induced domain.24,28 First, an illumination diaphragm was used so that only the center portion of the field of view was illuminated. Second, a manual light shutter was used to minimize unnecessary exposure of light to GUVs during experiments. Third, we also introduced a simple video microscopy technique; fluorescence images were continuously recorded in the video mode at a rate of 5 frame/second by the CCD camera during experiments. If lipid domains were observed on a GUV, we then examined the recorded video frame by frame to make sure that the domains were there right after the GUV was moved into the illuminated area. If the domains only appeared after a period of time after the GUV was moved into the illuminated area (for example, 0.5 seconds), these lipid domains must be light-induced domains.


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Measurement of The Emission Spectrum of Nile Red After trying out several fluorescence dyes (DPH-PC, di-pyrene-PC, …), we found that the emission spectrum of Nile Red is quite sensitive to lipid composition of liposomes and this property was used to determine the lipid compositions of coexisting lipid domains. Nile red is a watersoluble fluorescence dye. Its fluorescence, largely quenched in water, is intense in the hydrophobic region of lipid bilayers.29,30 Its emission spectrum shifts depending on the lipid composition. In our spectrum analysis, we ignored the background emission of Nile Red in water. Nile Red emission spectra were measured using a T-mode PTI C61/2000 spectrofluorimeter (Lawrenceville, NJ). The excitation wavelength was set to 530 nm with a 4-nm slit width. The emission spectra were collected between 560 to 710 nm with a 2-nm slit width. The emission signals from both detector channels were collected in the photon counting mode with a step size of 0.5 nm and 1 second integration time. For fluorescence spectrum measurements, RSE liposomes were labeled with 0.1 mol% of Nile Red fluorescence dye. 2 ml of RSE liposomes (~75 µM) suspension in aqueous buffer (5 mM PIPES/200 mM KCl/1 mM NaN3, pH 7.0) was added to a cuvette containing a Teflon coated magnetic stir bar. The cuvettes were kept at 25 C, the same temperature at which we performed GUV microscopy experiment. Measurement of Partition Coefficient of Gramicidin-A (Kp) Between Lo and Ld Phases We prepared multilamellar vesicles by the dry-film method following the procedure in literature.31,32 DOPC/DSPC/cholesterol lipid mixtures with or without 1 mol% gramicidin-A were first dissolved in chloroform. Samples were then dried to a thin film under a gentle stream of nitrogen while vortexing and then kept under vacuum for about 12 hours. The final pressure of the vacuum chamber was about 10 mTorr. Each sample was hydrated with 2.2 ml aqueous buffer for 1 hour under argon and vortexed for 30 seconds. The concentrations of suspension of multilamellar vesicles in aqueous buffer were about 75 µM. Hydration was done at 61C. At the end of hydration period, samples were cooled to room temperature at a rate ~5 C/hour. Fluorescence measurements were made with a T-mode PTI C61/2000 spectrofluorimeter (Lawrenceville, NJ) at 25 C using Quartz cuvettes. The excitation wavelength was set at 270 nm, and the peak of


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emission spectrum was 334 nm. The excitation and emission bandwidths were 2 and 3 nm, respectively. A 300 nm high-pass filter was used in the path of emission beam to cut down the scattering of excitation beam. Estimation of Fraction of Gramicidin not in Lipid Bilyers DOPC/DSPC/cholesterol/gramicidin-A liposome samples in aqueous buffer were prepared using the RSE method.26 Calibration samples of gramicidin in aqueous buffer (without lipids) at various concentrations were prepared using the same method. All samples were centrifuged at 15,000 g for 20 minutes and supernatants were collected. The gramicidin fluorescence emission spectrum (300 nm – 390 nm) was measured for each sample as described above. We found that about 15% of liposomes remained in supernatants after the centrifugation. Therefore, the fluorescence spectra of centrifuged samples were fitted to the spectra of uncentrifuged liposome sample and gramicidin-only calibration samples. From the fitting, the fraction of gramicidin not in lipid bilayers was estimated. RESULTS AND DISCUSSION Shifting of Lo+Ld Phase Boundary

We first validated our experimental techniques by

finding the Lo+Ld phase boundary of DOPC/DSPC/cholesterol ternary system, and compared it with the published phase boundary of the same system by Feigenson group.7, 33,34

We then prepared GUVs of DOPC/DSPC/cholesterol/gA quaternary mixtures. We

started with mixtures that have the same R value but different C values at 5 mol% increment in C. After locating the phase boundary approximately, we prepared and examined three independent sample sets at 2 mol% increment in C. By repeating this procedure for different R values with an accuracy of 2 mol% in C, we determined 16 boundary points (see Fig. 1). The phase boundary of Lo+Ld coexisting region was obtained by a polynomial fit to these 16 points.

Figure 1 shows the Lo+Ld phase

boundaries of DOPC/DSPC/cholesterol mixtures with and without 1 mol% of gA, together with images of GUVs containing 1 mol% of gA at selected lipid compositions.


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Above the phase boundary, a mixture has only one phase; below the phase boundary, a mixture has coexisting Lo and Ld phases.

Figure 1 Lo+Ld phase boundary of DOPC/DSPC/cholesterol mixtures with and without 1 mol% of gA and fluorescence images of GUVs containing 1 mol% of gA at some selected lipid compositions. The solid line is the phase boundary for DOPC/DSPC/cholesterol ternary mixtures;7,33,34 the dashed line is the phase boundary with 1 mol% of gA. Rigion I: the area between the two phase boundaries on the left side of the phase diagram; Rigion II: the area between the two phase boundaries on the right side. The scale bar in each GUV image represents 10 µm. Since DiIC12(12) fluorescent dye partitions favorably into the Ld phase, the bright domains are Ld phase domains and the dark domains are Lo domains.

Figure 1 shows that the phase boundary is tilted and shifted to the right after adding 1 mol% of gramicidin-A. The effect of adding gA on membrane’s phase behavior is quite complex and strongly depends on the lipid composition of the membrane. Region


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I in Figure 1 is a 2-phase region in the absence of gA; with gA, it becomes a one-phase (Ld) region. In contrast, Region II in the absence of gA has only one phase (Lo); but with gA, both Lo and Ld phases coexist in that region. However, at the majority lipid compositions in the 2-phase region, adding gramicidin changes the lipid compositions and amounts of the two coexisting phases. Indeed, quantitative description of these changes requires the knowledge of thermodynamic tie-lines and gA’s partition coefficients. Determination of Thermodynamic Tie-lines In the Lo+Ld 2-phase coexisting region, a thermodynamic tie-line is a straight line that starts from a point on the Ld side of the phase boundary and ends at a point on the Lo side. For a mixture with an overall lipid composition on the tie-line, the lipid compositions of coexisting Lo and Ld phases are given by the two end points, and the amount of each phase can be calculated by the Lever rule. A phase boundary together with thermodynamic tie-lines can give a complete description of coexisting Lo+Ld phases. Knowing the compositions of coexisting Lo+Ld phases is important for quantifying the effects of protein. Our method of determining tielines is based on the fact that the emission spectrum of Nile Red fluorescence continuously shifts to blue (i.e., shorter wavelength) along the phase boundary - from the Ld side to the Lo side (Figure 2). Applying the Lever-rule, the spectrum of a mixture in a 2-phase coexisting region is a linear superposition of the two single-phase spectra (Lo and Ld) at the two phase boundary points (i.e., two end points of a tie-line).


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Figure 2 Nile Red emission spectra along the Lo+Ld phase boundary and in the two-phase region. As composition of the lipid bilayer changes from point 1 (on the Ld border) to point 9 (on the Lo border), Nile Red spectrum exhibits a blue-shift. In order to see the shift clearly, we fitted the spectra by polynomials of 6 degree. The Gibbs’ triangle and 9 selected lipid compositions are shown in the graph.

We prepared RSE liposomes of quaternary DOPC/DSPC/cholesterol/gA mixtures labeled with Nile Red at the probe/lipid ratio of 1/1000. We selected 17 points on the Ld side of the boundary, and 15 points on the Lo side, as well as 3 other points in the twophase region on the R = 0.5 line with following lipid compositions: Point 1: DOPC/DSPC/Chol

=37.5/37.5/25

(i.e.,

R

=

0.5,

c

=

0.25),

Point

2:

DOPC/DSPC/Chol=35/35/30 (i.e., R = 0.5, c = 0.30), and Point 3: DOPC/DSPC/Chol =32.5/32.5/35 (i.e., R = 0.5, c = 0.35, see Figure 3b). Emission spectra of Nile Red were measured for all 35 samples at 25 C. For a given point inside the 2-phase region, all possible straight lines passing through that point were tested for spectrum fitting (Figure 3a). According to the Lever-rule, the spectrum of a mixture in the coexisting Ld+Lo region is a linear superposition of the spectra of two endpoints of the tie-line, Id() and


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Io(). Therefore, we define a fitting curve for the intensity of emission spectrum in the coexisting Ld+Lo region as Ifit() = A [fd Id() + (1- fd) Io()]

(1)

Where fd is the fraction of mixture in the Ld phase determined from the phase diagram, and A is an adjustable scaling factor. We define a fitting goodness parameter  as  710

   (I fit ()  I())2

(2)

 560

Where I() is the measured intensity of emission spectrum of Nile Red for a mixture in

 the coexisting Ld+Lo region. In order to find the tie-line for a given point on R=0.5 line in coexisting Ld+Lo region, we search for the minimum value of fitting goodness  by varying the scaling factor A numerically. Figure 3a shows all possible straight lines (trial tie-lines) tested and also the best-fit line (i.e, the tie-line, in red) for Point 3 in the coexisting Lo+Ld region. Figure 3b shows the three thermodynamic tie-lines determined using the Nile Red spectrum fitting method. The errors of tie-lines were estimated based on the fitting goodness parameter compared to the noise level in the Nile Red spectra, the experimental uncertainty in lipid compositions of the samples, and visual inspection of the fits. We confirmed the above tie-line results also using another fluorescent molecule, DPH-PE, and we got the very similar result. However, Nile Red spectra are more sensitive to the change of lipid composition; thus Nile Red is preferred.


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Figure 3 (a) The procedure for searching thermodynamic tie-line. Solid lines are all possible straight lines passing through Point 3 with lipid composition DOPC/DSPC/chol=32.5/32.5/35. Each straight line connects a point on the Ld side of the phase boundary to a point on the Lo side. The red line is the best-fit line (i.e, the tie-line) for Point 3. (b) Lo+Ld phase boundary and thermodynamic tie-lines for DOPC/DSPC/cholesterol mixtures with 1 mol% of gA.


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Figure 4 Nile Red emission spectra of the 2-phase mixture DOPC/DSPC/Chol=37.5/37.5/25 without gA (i.e., Point 1, red), Ld (green) and Lo (blue) end points of the determined tie-line, and the best-fit curve using Eq.1 (black).

We also measured the tie-lines passing through Points 1, 2, and 3 for the ternary mixtures without gA using the Nile Red method (the blue lines in Fig. 5). Figure 4 shows Nile Red emission spectra of the best-fit tie-line passing through Point 1; the fitting of spectrum is excellent. Our tie-lines agree well with those of Heberle et al. (2010) for the same system33. The differences in tie-line slopes from the two studies using two different methods are less than one degree. This validates our method of determining tie-lines using Nile Red emission spectra.


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Figure 5 Comparison of tie-lines passing through Points 1, 2 and 3. Blue lines: tie-lines without gA; Red lines: tie-lines with 1 mol% of gA. The squares at ends of tie-lines represent error bars in determining tie-lines. d2 and o2 are the lipid compositions of Ld and Lo phases for the tie-line passing through Point 2 without gA, respectively; d2* and o2* are the lipid compositions of Ld and Lo phases with 1 mol% of gA, respectively.

Quantitative Analysis of Effect of Adding Gramicidin

By comparing two sets of phase

boundaries and two sets of tie-lines, the effects of adding 1 mol% of gA to 2-phase mixtures of DOPC/DSPC/chol can be completely quantified. Figure 5 shows tie-lines for DOPC/DSPC/chol mixtures with and without gA passing through lipid composition Points 1, 2 and 3 in Figure 3b. Without gA, the tie-lines have smaller positive slopes; with gA, they have larger slopes, showing the effect of gramicidin. By applying the Lever-rule, these tie lines enable us to make a precise quantitative analysis. Table 1 lists


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the lipid compositions of coexisting phases with and without gA for the mixture with lipid composition of Point 2. d2 and o2 are lipid compositions of Ld and Lo domains without gramicidin, respectively; d2* and o2* are lipid compositions of Ld and Lo domains with 1 mol% of gramicidin, respectively. As expected, the dominating lipid species in Ld domains is unsaturated DOPC, and Lo domains are rich in saturated DSPC and cholesterol. By comparing the compositions of membrane domains d2, d2*, o2, and o2*, we conclude that gramicidin alters the lipid compositions of coexisting phases. Adding gramicidin decreases the mole fraction of cholesterol in Ld domains and increases cholesterol in Lo domains. Without gramicidin, the ratio of amounts of cholesterol in the Lo phase to that in the Ld phase is ~1.7; with gramicidin, the ratio increases to ~2.2. Furthermore, the R values for both Lo and Ld phases increase with gramicidin. The above quantitative analyses can be performed for any given point in the 2-phase region. Thus, two sets of phase boundaries together with two sets of tie-lines can be a powerful tool to quantitatively describe the effects of a membrane protein on coexisting lipid domains for any given mixture over the entire 2-phase region. As far as we know, this is the first study that gives precise changes of membrane domain compositions induced by a membrane protein. Table 1 Comparison of lipid compositions of coexisting Lo and Ld domains for 2-phase DOPC/DSPC/chol=35/35/30 lipid mixture (i.e., Point 2) with and without 1 mol% of gA.

Points in Figure 5

Lipid composition (DOPC/DSPC/cholesterol) 70/8.6/21.4 70.6/11/18.4 9.8/54/36.2 6.5/53.8/39.7

d2 (Ld domains without gA) d2* (Ld domains with gA) o2 (Lo domains without gA) o2* (Lo domains with gA)

Partition of Gramicidin-A Between Lo and Ld Phases We also measured gramicidin partition coefficients for two tie-lines passing through Point 2 and Point 3 in the Lo+Ld coexisting region. Partition coefficient, Kp, is the ratio of equilibrium gramicidin concentration in the Ld phase to that in the Lo phase. Figure 6a shows the emission


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spectrum of 1 mol% gramicidin-A in 74.4/10.6/15 DOPC/DSPC/Chol lipid mixture. For each tie-line, eleven lipid mixtures (all with 1 mol% gA) and eleven corresponding background samples (without gA) were prepared with compositions spanning the full length of the tie-lines. The fluorescence intensities of all samples were integrated for a period of 180 s at the peak of the emission spectrum, 334 nm (see Fig. 6a insert). The normalized fluorescence intensity for each tie-line was plotted as a function of the Lo phase fraction (fLo), which has the values of 1 and 0 at the Lo and Ld end points of the tieline, respectively. The fluorescence intensity data were fitted to Equation 335 (Figure 6b).

where FN(Ld) and FN(Lo) are the normalized fluorescence intensities at the Ld and Lo end points, respectively. From the fitting, the partition coefficients Kp were determined to be 3.70.3 and 1.70.2 for tie-lines passing through Point 2 and Point 3, respectively. The result indicates that gramicidin prefers the Ld phase over the Lo phase. As expected, partition coefficient is not a constant and it depends on the position of tie-line in the coexisting phase region. When a tie-line is very close to the critical point, the value of partition coefficient should approach unity, because two coexisting phases become nearly indistinguishable. Away from the critical point, such as on the tie-line passing through Point 2, the partition coefficient is 3.7, indicating that gramicidin prefers the disordered Ld lipid domains with smaller bilayer thickness.


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Figure 6 (a) Fluorescence emission spectrum of gA in DOPC/DSPC/Chol=74.4/10.6/15 lipid bilayer. Insert: Fluorescence intensities of gA at its emission peak (334 nm) as a function of time at the two end points of the tie-line passing through Point 2. (b) Normalized fluorescence peak intensities of gA vs. Lo phase fraction along tie-lines passing through Point 2 and Point 3. The data was fitted to Equation 3 to obtain the partition coefficient Kp and the fitting curves.

Dibble et al. measured partition coefficient of gramicidin in binary phospholipid mixtures containing coexisting fluid/gel phases using a fluorescence quenching method.31 The spin-labeled phosphatidylcholine, (7,6)PC, was used as the fluid-phase lipid, and Ca(di18:1PS)2, di18:0PC, di:16:0PC or di14:0PC was used as the gel-phase lipid. They found that gramicidin prefers the (7,6)PC-rich fluid-phase bilayer with small bilayer thickness, and the partition coefficient is ~30 for Ca(di18:1PS)2 and di18:0PC, ~10 for di16:0PC and ~ 1 for di14:0PC. The results show that when the hydrophobic mismatch between the length of gramicidin and the length of the phospholipid acyl chain increases, partitioning of gramicidin in gel phase decreases (i.e., Kp increases). Interestingly, the partitions of gramicidin in the (7,6)PC-rich fluid-phase bilayer and the gel-phase di14:0PC bilayer are nearly equal. Dibble et al. concluded that bilayer thicknesses, not the gel/fluid states of the bilayers, largely determine the partition of gramicidin.31 The partition coefficients we obtained are 3.7 and 1.7, which are significantly smaller than the values obtained in fluid/gel mixtures by Dibble et al.31 The partition coefficients measured in this study are between two fluid phases (Ld and Lo). The Lo phase is rich in


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DSPC and cholesterol, and Ld phase is rich in unsaturated DOPC (see Table 1). The Lo phase has tighter molecular packing and a larger bilayer thickness than Ld phase. Gramicidin is a short transmembrane peptide, and it prefers lipid bilayers with small bilayer thickness. A recent x-ray diffraction measurement of DOPC/DSPC/cholesterol Ld and Lo mixtures with lipid compositions very similar to our study showed that the bilayer thicknesses of Ld and Lo phases are approximately 3.9 and 4.9 nm, respectively.36 The bilayer thicknesses of both Ld and Lo domains in our system are likely larger than the hydrophobic length of a gramicidin channel. Although gramicidin still prefers the Ld domains with smaller bilayer thickness, it is quite likely that gramicidin’s partition in the Ld phase is not as favorable as that in the (7,6)PC-rich fluid-phase, explaining the low values of partition coefficient in our system. Ideally, the samples for the partition coefficient measurement should also be prepared by the RSE method. However, we found that gA fluorescence intensities are noisier from RSE samples than from dry film samples. One possible source of the noise could be the different conformations of membrane-bound gA (channel, nonchannel, monomer, intermediate I and intermediate II), which have different fluorescence characteristics (emission peak, intensity, …).37 We did not attempt to control gA conformation in our samples by modifying our GUV and RSE preparation procedures, which may not be a trivial task by itself. As pointed out by Dibble et al., the hydrophobic mismatch would still occur between a gramicidin monomer and a single leaflet of the bilayer.31 Although there could be some differences between the partition of gA in RSE vesicles and that in multilamellar vesicles, we believe that the general conclusion that gA prefers thinner Ld domains still hold. The fraction of gramicidin not in lipid bilayers Although we kept the mole fraction of gramicidin at 1 %, it is possible that with our sample preparation method, a small fraction of gramicidin partition into the aqueous phase, instead of lipid bilayers. Based on an external surface area assay, it was determined that most liposomes prepared by the RSE method have one or two lipid bilayers;38 so RSE liposomes are difficult to be completely


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removed by centrifugation. The peak of gA emission spectrum in liposomes is at ~334 nm, and the peak of gA emission in buffer (without liposome) is at ~340 nm. After the 20 minutes centrifugation at 15,000g, we estimated that about 15% of liposomes remained in supernatants. It is reasonable to assume that the remaining liposomes are mostly small and unilamelar. Thus, the fluorescence spectrum of supernatant is a combination of spectra of gramicidin in small liposomes and in buffer. Via spectrum fitting, we determined that the amount of gramicidin in aqueous phase was between 2% to 5% of the total gramicidin. Thus, 95% to 98% of gramicidin was in lipid bilayers. Gramicidin has many hydrophobic residues and low solubility in water; our estimate seems reasonable. The general direction of Membrane composition changes by gramicidin

Membrane

domain compositional changes shown in Table 1 result from the molecular interactions between gramicidin and lipid bilayers. Previous studies of gramicidin have shown that a key interaction between gramicidin channels and lipid bilayers is the hydrophobic mismatch – the bilayer adjacent to a gramicidin channel changes its thickness to match the hydrophobic length of gA channel. The magnitude of this protein-lipid interaction is likely larger than that of typical lipid-lipid interaction, because it causes significant structural changes to lipid bilayers, in addition to the fact that a typical protein occupies a larger area than a lipid. Our data clearly shows that gramicidin peptides alter the overall lipid compositions of the two types of coexisting membrane domains.

It will be

interesting to ask: what causes the changes in lipid composition and can we predict the changes in other bilayer mixtures? It has been shown that the hydrophobic length of a gramicidin channel matches well with a lipid bilayer composed of lipids with 14 carbons in their acyl chains (such as DMPC31). Since we do not measure bilayer thickness in this study, we will estimate the changes of bilayer thicknesses based on the changes of bilayer lipid compositions. Both DOPC and DSPC have 18 carbon chains, and cholesterol is known to increase the bilayer thicknesses of fluid-phase lipid bilayers.39 Therefore, the bilayer thickness of Ld domain should be larger than the hydrophobic length of gramicidin channels, and Lo domains are even thicker.25,36 As shown in Table 1, gramicidin decreases cholesterol in Ld domains and increase cholesterol in Ld domains,


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which should enlarge the bilayer thickness difference between Lo and Ld domains.39,40 Consequently, it will drive more gramicidin into the thinner Ld domains. Furthermore, a number of studies have shown that cholesterol decreases gramicidin channel activity.41-43 Therefore, decreasing cholesterol in Ld domains should assist the function of gramicidin. Thus, the changes in cholesterol mole fraction induced by gramicidin facilitate the partition and function of gramicidin in Ld domains. The increasing of R values in both Lo and Ld phases may increase the bilayer thicknesses slightly for both types of domains. However, it may have little effect on gramicidin’s partition preference. It is quite likely that in addition to produce changes in overall bilayer properties, gramicidin channels also create local perturbation of bilayer thickness to further reduce the free energy. Based on our data, we propose that the general result of gramicidin-lipid interaction in a membrane containing coexisting phases is: the lipid compositions of membrane domains are changed in such way that favors the partition and function of gramicidin channels. More studies of this kind using different multi-phase bilayer systems are needed to test this hypothesis. Membrane protein concentration

In our experiments, we limited gramicidin

concentration to 1 mol%, because higher concentration of gramicidin could transfer lipid bilayers into reverse hexagonal (HII) phase.44 Assume that the cross-sectional area of a gramicidin

channel

is

about

3.2

nm2

and

average

area-per-lipid

for

DOPC/DSPC/Cholesterol mixtures is about 0.42 nm2,25 at 1 mol% of gramicidin, about 7% of bilayer area is occupied by gramicidin. Since more gramicidin peptides are present in the Ld domains, the bilayer area occupied by gramicidin in Ld domains could be as high as 11% in our experiments. Biomembranes are crowded with proteins. For human red blood cell plasma membrane, it has been estimated that proteins cover 23% of membrane area.10 In other membranes, the estimations go as high as 50%.11 Our data show that at 7% of membrane area, gramicidin already induces significant global changes to membrane domains: altering overall lipid compositions and bilayer thicknesses. These changes reflect fundamental protein-lipid interactions. At a higher protein concentration, greater effects are expected. It will be very interesting to find out what will be the sizes


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and structure of membrane domains at physiological protein concentrations. It is possible that proteins at high concentration could abolish macroscopic phases, as demonstrated previously in a Monte Carlo simulation.45 If membrane domains become nanoscopic, instead of optical microscopy, other experimental techniques such as FRET and neutron scattering can be used to characterize membrane nanodomains.46 CONCLUSIONS In DOPC/DSPC/Cholesterol ternary mixtures containing coexisting Lo and Ld phases, gramicidin as an active component of lipid membranes, not only partitions favorably into the thinner Ld phase, but also alters the phase boundary and thermodynamic tie-lines. Even at as low as 1 mol% (i.e., ~7% of membrane area), gramicidin decreases the cholesterol mole fraction of the Ld domains, and opposite effect is produced in the Lo domains. These compositional changes result in thinner Ld domains and thicker Lo domains, which facilitates the preferential partition of gramicidin into the thinner Ld domains. This study shows that trans-membrane proteins have significant contribution to the overall lipid composition of membrane domains. Membrane domain size, composition, order, and protein partition behavior are important for biomembrane functions, membrane protein activity, disease development and diagnosis, and drug design. This study will advance our knowledge about biomembranes and protein-lipid interactions.


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REFERENCES: (1) Engelman, D. M. Membranes are more mosaic than fluid. Nature 2005, 438 (7068), 57880. (2) Owen, D. M.; Williamson, D. J.; Magenau, A.; Gaus, K. Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution. Nat Commun 2012, 3, 1256. (3) Brown, D. A.; London, E. Functions of lipid rafts in biological membranes. Annual Review of Cell and Developmental Biology 1998, 14, 111-136. (4) Brown, D. A.; London, E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. Journal of Biological Chemistry 2000, 275 (23), 17221-17224. (5) Simons, K.; Ikonen, E. Functional rafts in cell membranes. Nature 1997, 387 (6633), 569572. (6) Feigenson, G. W. Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures. Biochim Biophys Acta 2009, 1788 (1), 47-52. (7) Konyakhina, T. M.; Wu, J.; Mastroianni, J. D.; Heberle, F. A.; Feigenson, G. W. Phase diagram of a 4-component lipid mixture: DSPC/DOPC/POPC/chol. Biochimica Et Biophysica ActaBiomembranes 2013, 1828 (9), 2204-2214. (8) Veatch, S. L.; Gawrisch, K.; Keller, S. L. Closed-loop miscibility gap and quantitative tielines in ternary membranes containing diphytanoyl PC. Biophys J 2006, 90 (12), 4428-36. (9) Veatch, S. L.; Keller, S. L. Seeing spots: Complex phase behavior in simple membranes. Biochimica Et Biophysica Acta-Molecular Cell Research 2005, 1746 (3), 172-185. (10) Dupuy, A. D.; Engelman, D. M. Protein area occupancy at the center of the red blood cell membrane. Proc Natl Acad Sci U S A 2008, 105 (8), 2848-52. (11) Luckey, M. Membrane Structural Biology: With Biochemical and Biophysical Foundations; 2nd ed.; Cambridge University Press: New York, 2014. (12) Mitra, K.; Ubarretxena-Belandia, I.; Taguchi, T.; Warren, G.; Engelman, D. M. Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol. Proc Natl Acad Sci U S A 2004, 101 (12), 4083-8. (13) Dowhan, W.; Bogdanov, M. Lipid-dependent membrane protein topogenesis. Annu Rev Biochem 2009, 78, 515-40. (14) Simons, K.; Gerl, M. J. Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 2010, 11 (10), 688-99. (15) Andersen, O. S.; Koeppe, R. E., 2nd. Bilayer thickness and membrane protein function: an energetic perspective. Annu Rev Biophys Biomol Struct 2007, 36, 107-30. (16) de Planque, M. R.; Greathouse, D. V.; Koeppe, R. E., 2nd; Schafer, H.; Marsh, D.; Killian, J. A. Influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers. A 2H NMR and ESR study using designed transmembrane alpha-helical peptides and gramicidin A. Biochemistry 1998, 37 (26), 9333-45. (17) Dumas, F.; Tocanne, J. F.; Leblanc, G.; Lebrun, M. C. Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport. Biochemistry 2000, 39 (16), 4846-54. (18) Killian, J. A. Hydrophobic mismatch between proteins and lipids in membranes. Biochim Biophys Acta 1998, 1376 (3), 401-15.


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(19) Lin, Q.; London, E. Altering hydrophobic sequence lengths shows that hydrophobic mismatch controls affinity for ordered lipid domains (rafts) in the multitransmembrane strand protein perfringolysin O. J Biol Chem 2013, 288 (2), 1340-52. (20) Lundbaek, J. A.; Collingwood, S. A.; Ingolfsson, H. I.; Kapoor, R.; Andersen, O. S. Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes. J R Soc Interface 2010, 7 (44), 373-95. (21) Ren, J.; Lew, S.; Wang, J.; London, E. Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length. Biochemistry 1999, 38 (18), 5905-12. (22) Martinac, B.; Hamill, O. P. Gramicidin A channels switch between stretch activation and stretch inactivation depending on bilayer thickness. Proc Natl Acad Sci U S A 2002, 99 (7), 430812. (23) Kingsley, P. B.; Feigenson, G. W. The synthesis of a perdeuterated phospholipid: 1,2dimyristoyl-sn-glycero-3-phosphocholine-d72. Chem. Phys. Lipids 1979, 24 (2), 135-147. (24) Baykal-Caglar, E.; Hassan-Zadeh, E.; Saremi, B.; Huang, J. Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity. Biochim Biophys Acta 2012, 1818 (11), 2598-604. (25) Hassan-Zadeh, E.; Baykal-Caglar, E.; Alwarawrah, M.; Huang, J. Complex roles of hybrid lipids in the composition, order, and size of lipid membrane domains. Langmuir 2014, 30 (5), 1361-9. (26) Buboltz, J. T. A more efficient device for preparing model-membrane liposomes by the rapid solvent exchange method. Rev Sci Instrum 2009, 80 (12), 124301. (27) Angelova, M. I.; Soleau, S.; Meleard, P.; Faucon, J. F.; Bothorel, P. Preparation of Giant Vesicles by External Ac Electric-Fields - Kinetics and Applications. Prog Coll Pol Sci S 1992, 89, 127-131. (28) Ayuyan, A. G.; Cohen, F. S. Lipid peroxides promote large rafts: Effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation. Biophysical Journal 2006, 91 (6), 2172-2183. (29) Fowler, S. D.; Greenspan, P. Application of Nile Red, a Fluorescent Hydrophobic Probe, for the Detection of Neutral Lipid Deposits in Tissue-Sections - Comparison with Oil Red O. J Histochem Cytochem 1985, 33 (8), 833-836. (30) Genicot, G.; Leroy, J. L. M. R.; Van Soom, A.; Donnay, I. The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes. Theriogenology 2005, 63 (4), 1181-1194. (31) Dibble, A. R.; Yeager, M. D.; Feigenson, G. W. Partitioning of gramicidin A' between coexisting fluid and gel phospholipid phases. Biochim Biophys Acta 1993, 1153 (2), 155-62. (32) Rawat, S. S.; Kelkar, D. A.; Chattopadhyay, A. Monitoring gramicidin conformations in membranes: A fluorescence approach. Biophysical Journal 2004, 87 (2), 831-843. (33) Heberle, F. A.; Wu, J.; Goh, S. L.; Petruzielo, R. S.; Feigenson, G. W. Comparison of three ternary lipid bilayer mixtures: FRET and ESR reveal nanodomains. Biophys J 2010, 99 (10), 330918. (34) Zhao, J.; Wu, J.; Heberle, F. A.; Mills, T. T.; Klawitter, P.; Huang, G.; Costanza, G.; Feigenson, G. W. Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/cholesterol. Biochimica Et Biophysica Acta-Biomembranes 2007, 1768 (11), 27642776.


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(35) Silvius, J. R. Partitioning of membrane molecules between raft and non-raft domains: insights from model-membrane studies. Biochim Biophys Acta 2005, 1746 (3), 193-202. (36) Heftberger, P.; Kollmitzer, B.; Rieder, A. A.; Amenitsch, H.; Pabst, G. In situ determination of structure and fluctuations of coexisting fluid membrane domains. Biophys J 2015, 108 (4), 854-62. (37) Rawat, S. S.; Kelkar, D. A.; Chattopadhyay, A. Monitoring gramicidin conformations in membranes: a fluorescence approach. Biophys J 2004, 87 (2), 831-43. (38) Buboltz, J. T.; Feigenson, G. W. A novel strategy for the preparation of liposomes: rapid solvent exchange. Biochim Biophys Acta 1999, 1417 (2), 232-45. (39) Alwarawrah, M.; Dai, J.; Huang, J. A molecular view of the cholesterol condensing effect in DOPC lipid bilayers. J Phys Chem B 2010, 114 (22), 7516-23. (40) Dai, J.; Alwarawrah, M.; Huang, J. Instability of cholesterol clusters in lipid bilayers and the cholesterol's Umbrella effect. J Phys Chem B 2010, 114 (2), 840-8. (41) Lundbaek, J. A.; Birn, P.; Girshman, J.; Hansen, A. J.; Andersen, O. S. Membrane stiffness and channel function. Biochemistry 1996, 35 (12), 3825-30. (42) Schagina, L. V.; Blasko, K.; Grinfeldt, A. E.; Korchev, Y. E.; Lev, A. A. Cholesteroldependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes. Biochim Biophys Acta 1989, 978 (1), 145-50. (43) Schagina, L. V.; Korchev, Y. E.; Grinfeldt, A. E.; Lev, A. A.; Blasto, K. Sterol specific inactivation of gramicidin A induced membrane cation permeability. Biochim Biophys Acta 1992, 1109 (1), 91-6. (44) Chupin, V.; Killian, J. A.; de Kruijff, B. 2H-nuclear magnetic resonance investigations on phospholipid acyl chain order and dynamics in the gramicidin-induced hexagonal HII phase. Biophys J 1987, 51 (3), 395-405. (45) Yethiraj, A.; Weisshaar, J. C. Why are lipid rafts not observed in vivo? Biophys J 2007, 93 (9), 3113-9. (46) Heberle, F. A.; Doktorova, M.; Goh, S. L.; Standaert, R. F.; Katsaras, J.; Feigenson, G. W. Hybrid and Nonhybrid Lipids Exert Common Effects on Membrane Raft Size and Morphology. Journal of the American Chemical Society 2013, 135 (40), 14932-14935.


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TOC


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0

1

Cholesterol

DOPC 0.5

0.5

Ld + Lo Region I

Region II

1

0 0

0.5

DSPC

ACS Paragon Plus Environment

1

Langmuir

Poly. (1)

1

Poly. (2)

0.9

Poly. (3) Poly. (4)

0.8

Poly. (5) Poly. (6)

0.7

Intensity (a.u.)

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

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Poly. (7) Poly. (8)

0.6

Poly. (9)

0.5 DOPC

0.4

(3)

0.3

(4)

Cholesterol

(7) (8)

(5)

(9) (2)

0.2

(6)

(1)

0.1 DSPC

0 560

580

600

620

640

λ (nm)


660

680

700

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a

1

0

DOPC

Cholesterol Point 3

0.5

0.5

1

0 0.5

0

1

DSPC

b

1

0

Cholesterol

DOPC 0.5

0.5

Point 3

Point 2 Point 1 1

0 0

0.5

DSPC

1


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 ACS Paragon Plus Environment

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0

DOPC

1

Point 3

Point 2

0.5

Cholesterol 0.5

o2*

o2 o2

d2 Point 1

d2* 1

0 0

0.5

DSPC


1

Langmuir

a

b

Intensity (a.u.)

Point 2 KP = 3.2 ± 0.3

80 60

Lo

0

60

40

120

Time (s)

Point 2 KP = 3.7± 0.3

1

Ld

180

20

Normalized Fluoresence (a.u.)

100

Intensity (a. u.)

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

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0.8 0.6

0.4

Point 3 KP = 1.7± 0.2

0.2 0

0 300

325

350

375

400

425

450

λ (nm)


0

0.2

0.4

0.6

Lo Fraction

0.8

1

Gramicidin Peptides Alter Global Lipid Compositions and Bilayer

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