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Co-spreading of anionic phospholipids with peptides of the structure (KX)4K at the air-water interface: Influence of lipid headgroup structure and hydrophobicity of the peptide on monolayer behavior. André Hädicke, Christian Schwieger, and Alfred Blume Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02255 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017

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Co-spreading of anionic phospholipids with peptides of the structure (KX)4K at the air-water interface: Influence of lipid headgroup structure and hydrophobicity of the peptide on monolayer behavior.

André Hädicke, Christian Schwieger and Alfred Blume* Institute of Chemistry, MLU Halle-Wittenberg, von-Danckelmann-Platz 4, 06120 Halle/Saale, Germany *Corresponding author. Address: Institute of Chemistry–Physical Chemistry, Martin-LutherUniversity Halle-Wittenberg, von-Danckelmann-Platz 4, D-06120 Halle/Saale, Germany. Tel: +49 345 55 25850 Fax: +49 345 55 27157 E-mail address: [email protected] Keywords: peptide-membrane binding, electrostatic interactions, hydrophobic interactions, lipid monolayers, epifluorescence microscopy, IRRAS

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Abstract Mixtures of anionic phospholipids (PG, PA, PS, and CL) with cationic peptides were cospread from a common organic solvent at the air-water interface. The compression of the mixed film was combined with epifluorescence microscopy or infrared reflection adsorption spectroscopy (IRRAS) to gain information on the interactions of the peptide with the different lipids. To evaluate the influence of the amino acid X of peptides with the sequence (KX)4K on the binding, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was mixed with different peptides with increasing hydrophobicity of the uncharged amino acid X. The monolayer isotherms of DPPG/(KX)4K mixtures show an increased area for the lift-off due to incorporation of the peptide into the liquid-expanded (LE) state of the lipid. The surface pressure for the transition from LE to liquid-condensed (LC) state is slightly increased for peptides with amino acids X with moderate hydrophobicity. For the most hydrophobic peptide (KL)4K two plateaus are seen at a charge ratio PG to K of 5:1 and a strongly increased transition pressure is observed for a charge ratio of 1:1. Epifluorescence microscopy images and infrared spectroscopy show that the lower plateau corresponds to the LE-LC phase transition of the lipid. The upper plateau is connected with a squeeze-out of the peptide into the subphase. To test the influence of the lipid headgroup structure on peptide binding (KL)4K was co-spread with different anionic phospholipids. The shift of the isotherm to larger areas for lift-off and to higher surface pressure for the LE-LC phase transition was observed for all tested anionic lipids. Epifluorescence microscopy reveals the formation of LC-domains with extended filaments indicating a decrease in line tension due to accumulation of the peptides at the LC-domain boundaries. This effect depends on the size of the headgroup of the anionic phospholipid.


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Introduction In all classes of life potent antimicrobial peptides (AMPs) or host defense peptides can be found as part of the innate immune system. The most important feature of AMPs is their cationic and mostly amphipathic character.1-2 The binding of these peptides to cell surfaces or lipid membranes is driven by electrostatic and hydrophobic interactions.3-6 The non-specific mode of action of these peptides is a key to circumvent the occurrence of resistance in bacteria. The lipid to peptide ratio and the lipid composition of the membranes are more decisive for the mode of action of the AMPs7-8 than their secondary structure, ranging from unordered,9 over β–sheets10-11 to α-helical.12-13 AMPs preferentially interact with negatively charged lipid membranes because of their cationic nature. Bacterial cell membranes contain a high proportion of negatively charged lipids such as phosphatidylglycerol (PG) or cardiolipin (CL) besides the zwitterionic phosphatidylethanolamines (PE).14-15 In contrast, mammalian cell membranes are composed mainly of zwitterionic lipids like phosphatidylcholine (PC), phosphatidylethanolamine, sphingomyelin (Sph) and sterols, such as cholesterol (Chol). Negatively charged components such as phosphatidic acid (PA) and phosphatidylserine (PS) are of minor abundance and mainly occur in the inner leaflet of the plasma membrane.16 Since the complexity of bacterial membranes is too high, model membranes containing negatively charged phospholipids are often used to study interactions with peptides. For investigations of surface binding, also lipid monolayers at the air-water interface are suitable. These monolayers represent half of a lipid bilayer membrane17-19 and are therefore a convenient model system for the investigation of interactions with peptides20-23 or other amphiphilic molecules.24-25 In monolayer experiments, the change in molecular area or surface pressure can easily be measured and controlled. To gain further information on the lipid phase state and the peptide arrangement the monolayer technique can be coupled with


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fluorescence microscopy17,

26-27

, Brewster-angle

microscopy,28-29 infrared reflection

absorption spectroscopy (IRRAS),30-32 or grazing incidence x-ray diffraction (GIXD).33-34 Previously, we have studied the interaction of a certain class of cationic peptides composed of lysine (K) and an uncharged amino acid with the general structure (KX)4K (X = glycine (G), alanine (A), α-amino butyric acid (Abu), valine (V), and leucine (L)) with lipid monolayers and bilayers.35-38 For the investigation of the interactions of peptides with lipid monolayers, two different experimental approaches can be applied: a) adsorption experiments, where a peptide solution is injected into the subphase underneath a preformed lipid monolayer at the air-water interface to form a mixed film, and b) co-spreading experiments, where both components are dissolved in an organic solvent and spread at the air-water interface to form instantly a mixed monolayer. In both cases, a compression of the monolayer yields the surface pressure-area isotherm of the mixed film. In the adsorption experiments, the initial surface pressure of the monolayer can be changed. The problem is that the adsorption process usually takes a very long time even with stirring of the subphase, so that it is not clear in all cases whether equilibrium is reached. In addition, stirring the subphase causes turbulences at the air-water interface so that epifluorescence fluorescence microscopy or Brewster angle microscopy cannot be used to determine domain structures. In co-spread monolayers, in a similar way, the peptides might also not be in equilibrium with the subphase, because the desorption process might be slow as well. In this paper we present surface pressure-area isotherms of co-spread mixtures of lipids and different amounts of peptides to determine the phase behavior at the air-water interface upon film compression. The combination of monolayer experiments with epifluorescence microscopy allowed us to visualize the phase transition process, where a separation into liquid expanded (LE) state and liquid condensed (LC) domains occurs. Moreover, morphological changes of the domains during film compression can be monitored. Infrared reflection


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absorption spectroscopy (IRRAS) was used to study the behavior of the lipids under compression, as the wavenumber of the methylene stretching vibrations of the acyl chains yields information on their conformation. Different types of negatively charged phospholipids were used to determine the influence of the chemical nature of the lipid headgroups.

Experimental Materials The lipids 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-dimyristoylsn-glycero-3-phosphatidic acid (DMPA) were purchased from Genzyme Pharmaceuticals LLC (Liestal, Switzerland). 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2dipalmitoyl-sn-glycero-3-phosphoglycerol

(DPPG),

and

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

phosphocholine (DMPC) were products from Lipoid GmbH (Ludwigshafen, Germany). 1,2Dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) and 1,1',2,2'-tetramyristoyl-cardiolipin (TMCL) were purchased from Avanti Polar Lipids Inc. (Alabaster, USA). 1,2Dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh–DHPE) was obtained from Invitrogen (Karlsruhe, Germany). The peptides (KX)4K with X = G, A, Abu, V, and L were custom-made via Fmoc solid phase chemistry with a purity above 98 % by GeneCust Europe (Dudelange, Luxemburg). Chloroform, methanol (HPLC grade) and NaCl were purchased from Carl Roth GmbH&CO KG (Karlsruhe, Germany). All chemicals were used as received without further purification. Aqueous solutions were prepared with ultrapure water from a Milli-Q Advantage A10 system (Millipore S.A.S., Molsheim Cédex, France). Conductivity was lower than 0.055 µS/cm (25 °C) and TOC below 5 ppb.


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Methods Monolayer measurements coupled with epifluorescence microscopy Fluorescence microscopy imaging of monolayers at the air-water interface was performed using an Axio Scope A1 Vario epifluorescence microscope (Carl Zeiss MicroImaging, Jena, Germany). Underneath the microscope a Langmuir Teflon trough with a maximal area of 20.8 cm2 and a moveable computer-controlled Teflon barrier (Riegler & Kirstein, Berlin, Germany) was positioned on an x–y-z stage (Märzhäuser, Wetzlar, Germany) to be able to move the film surface with respect to the objective lens to any desired position. The x–y–z motion control was managed by a MAC5000 system (Ludl Electronic Products, Hawthorne, NY, USA). The trough was enclosed by a home-built Plexiglas hood to ensure a dust-free environment and to minimize evaporation of water. The temperature of 20.0 ± 0.1 °C was maintained with a circulating water bath and the whole setup was placed on a vibrationdamped optical table (Newport, Darmstadt, Germany). The air-water surface was illuminated using the following set up from Carl Zeiss MicroImaging (Jena, Germany): a 100 W mercury arc lamp (HXP 120 C), a long working distance objective (LD EC Epiplan-NEOFLUAR 50x) and a filter/beam splitter combination (Zeiss Filter Set 81HE), to select appropriate wavelengths for the excitation and detection of the Rh–DHPE fluorescence. Images were recorded using an EMCCD camera (ImageEM C9100-13, Hamamatsu, Herrsching, Germany). Image analysis and data acquisition were done using the AxioVision software (Carl Zeiss MicroImaging, Jena, Germany). All presented images show areas of individually contrast-adjusted raw data. The lipid solutions with a concentration of 1 mM lipid in chloroform contained only 0.01 mol% fluorescently labelled Rh–DHPE. This low fluorescence label concentration was possible due to the high quantum yield of Rh–DHPE and the high sensitivity of our CCD camera. Rh–DHPE prefers the liquid-expanded state of a lipid monolayer so that liquid-condensed domains appear dark.35, 39 The peptides were dissolved in methanol. Lipid and peptide solutions were mixed prior to the experiment to gain the desired ACS Paragon Plus Environment

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charge ratio of lipid to peptide. Mixed monolayers were prepared by spreading this stock solution onto the water surface. The subphase contained 100 mM NaCl. After waiting for at least 15 min to ensure complete solvent evaporation, the monolayer was compressed with a compression speed of 2 Å2·lipid molecule-1·min-1. Microscopy images were taken during the compression of the monolayer at various surface pressures. Fourier Transformed-Infrared Reflection Absorption Spectroscopy (FT-IRRAS) FT-IRRAS was performed using a BRUKER Vector 70 FT-IR spectrometer equipped with a nitrogen cooled MCT detector and an A511 reflection unit (Bruker Optics, Germany) containing the Langmuir trough setup (Riegler & Kirstein, Germany). A trough with two compartments in a hermetically sealed box was used. The sample trough (length: 30 cm, width: 6 cm, height 0.3 cm) was equipped with a Wilhelmy plate as pressure sensor. The circular reference trough (diameter 6 cm, height 0.3 cm) placed next to the sample trough can be brought into the focus of the IR beam by means of a trough shuttle. The temperature of 20 ± 0.1 °C was maintained using a circulating water bath. The filling levels of both troughs were kept equal and constant by means of an automated, laser reflection controlled pumping system, connected to reservoirs of purified, deionized water. P-polarized IR light obtained with a KRS-5 wire-grid polarizer was focused onto the water surface at a fixed angle of incidence ϕ = 40° with respect to the surface normal. Reflectance-absorbance spectra (RA) were calculated from the single-beam reflectance spectra recorded on the reference (R0) and sample trough (R) according to RA = -lg(R/R0). Resolution and scanner speed were set to 4 cm-1 and 80 kHz, respectively, for all experiments. 1000 scans were accumulated for measurements using p-polarized light. Zero filling by a factor of two and Blackman-Harris apodization were applied to the averaged interferograms before Fourier transformation yielding the spectra R and R0.


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Lipid (1mM in chloroform) and peptide (in methanol) solutions were mixed prior to the experiment to gain the desired lipid to peptide charge ratio. Mixed monolayer films were prepared by spreading this stock solution onto the water surface. The subphase contained 100 mM NaCl. After waiting for complete solvent evaporation and water vapour equilibration, compression of the monolayer and measurements of IRRA-spectra were started simultaneously. The monolayer was compressed with a speed of 2 Å2·lipid molecule-1·min-1. A reference reflectivity spectrum was recorded before each sample spectrum. The subphase level was adjusted before each data acquisition. All IRRA-spectra were corrected for atmospheric contribution (H2O vapor and CO2) and shifted to a zero baseline in a spectral region where no vibrational peak occurred (2800 cm-1 – 2750 cm-1). To determine the position of the vibrational bands in a certain wavenumber interval, second derivatives of spectra were calculated and the ‘peak picking’ function included in the Bruker OPUS software was used.

Results and Discussion All the used negatively charged lipids carry in the headgroup region their negative charge, which is delocalized over the phosphate group. The positive charge of the peptide is located at the terminal amine group in the side chain of the lysine residue. The assumed electrostatic interactions preferentially occur between these two moieties. Furthermore, hydrophobic interactions driving the binding of the peptide to the lipid monolayer are found between the lipid acyl chains and the side chains of the uncharged side chain residues of the peptide. The extent of this effect can be tuned by the size of the hydrophobic side chain of the peptide. Mixtures of DPPG with different (KX)4K peptides and at different charge ratios Rc were prepared to test for the influence of the peptides on the monolayer behavior. The charge ratio Rc, defined as [lipid]/[K] was changed from 20:1 up to 1:1, i.e. complete charge compensation. As each individual peptide contains 5 positive charges, the lipid to peptide ACS Paragon Plus Environment

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ratio is five-fold higher than the charge ratio. The monolayer compression experiments were coupled with epifluorescence microscopy and infrared reflection absorption spectroscopy to investigate the effect of the co-spread peptide on the domain formation process, the domain shapes and the ordering in the acyl chain region. Pure DPPG The isotherm of pure DPPG spread onto a 100 mM NaCl solution shows a lift-off from 0 mN m-1 at a molecular area of about 115 Å2 molecule-1 (see Figure 1). Further compression of the LE state monolayer increases the surface pressure up to Π = 9.4 mN m-1, where the phase transition into the LC state occurs. The observed values correspond well with DPPG isotherms reported in literature.40-43 Co-spreading of DPPG with (KX)4K peptides DPPG/(KG)4K (KG)4K is the least hydrophobic of the tested (KX)4K peptides. The mixed monolayer composed of DPPG and (KG)4K with a charge ratio of 2:1 (equivalent to a lipid to peptide ratio of 10:1) shows the same area for the lift-off (115 Å2 molecule-1) and the same behavior in the LE state (see Figure 1a). The LE-LC phase transition remains unchanged at a surface pressure of 9.4 mN m-1. At the end of the transition plateau, a slight difference of the isotherm of the mixed monolayer compared to the one of pure DPPG is seen, namely a steeper surface pressure increase at an area of 55 Å2 molecule-1. At high surface pressure (Π > 20 mN m-1) both isotherms are identical. At a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1), the lift-off is shifted to a slightly higher area per molecule (120 Å2 molecule-1). As already discussed in the adsorption studies35, the interaction between the lipids and the peptides at the air-water interface is an overlay of electrostatic and hydrophobic contributions. A net increase in area could correspond to a situation where the lipid area is decreased due to


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a decrease in electrostatic repulsion resulting from charge neutralization by the peptide. The concomitant incorporation of the peptide into the monolayer caused by hydrophobic interactions increases the total area. Apparently, the peptide molecules adsorb to the air-water interface coexisting with DPPG molecules in the LE state. However, this adsorption does not occur when the peptide is injected into the subphase underneath a pure air-water interface without lipids. Thus, adsorption is apparently driven by interactions with the lipids at the interface. However, the presence of (KG)4K has only minor influence on the LE-LC phase transition pressure of DPPG, which occurs at 9.6 mN m-1 in the mixed film, i.e. only slightly higher than in pure DPPG monolayers. For values of Π > 30 mN m-1 the isotherm is similar to those for pure DPPG, suggesting a squeeze-out of the peptide into the subphase.


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Π-A isotherms of co-spread mixtures of DPPG with (KX)4K peptides with different charge ratios as indicated: a) DPPG/(KG)4K, b) DPPG/(KA)4K, c) DPPG/(KAbu)4K, d) DPPG/(KV)4K, e) DPPG/(KL)4K. For comparison, the isotherm of pure DPPG is also shown. The aqueous subphase always contained 100 mM NaCl. The area is given as area per lipid molecule f) LE-LC phase transition pressures as a function of 1/Rc for the different lipidpeptide mixtures. As the two plateau regions are hard to see by eye, we added the compressibility graphs of the mixed films in the Supporting Information (see Figure S1) to prove the existence of the 2 plateau regions.


1.1

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DPPG/(KA)4K The compression isotherms of co-spread mixtures of (KA)4K with DPPG (see Figure 1b) show a similar behavior as observed for mixtures of DPPG with (KG)4K. The lift-off is shifted to larger areas per lipid for both tested charge ratios. The LE-LC phase transition occurs for Rc = 1:1 (equivalent to a lipid to peptide ratio of 5:1) at a slightly higher surface pressure and the plateau region is shifted to larger areas per lipid compared to the isotherm of pure DPPG. Also, the end of the transition plateaus is less defined in the isotherms of the mixed monolayers, i.e. the transition is less cooperative. Calculation of the compressibility profiles of the mixed monolayers reveals the occurrence of a second transition pressure at 10.4 mN m-1 for mixtures with Rc = 1:1 mixtures (see Figure 1f). The origin for this second transition remains unresolved. In the LC state, the isotherm shows some deviation at surface pressure values Π = 20 – 35 mN m-1 due to interaction of the peptide with the LC-domains via mainly electrostatic contributions. At Π > 35 mN m-1 the isotherm converges with that of pure DPPG, again indicating the squeeze-out of the peptide into the subphase. DPPG/(KAbu)4K Isotherms of co-spread DPPG/(KAbu)4K mixtures of the tested charge ratios of 20:1 (equivalent to a lipid to peptide ratio of 100:1) and 2:1 (equivalent to a lipid to peptide ratio of 10:1) are showing comparable behavior as that discussed before for the more hydrophilic peptides (KG)4K and (KA)4K. Only for Rc = 1:1 a larger shift of the lift-off value to 130 Å2 molecule-1 is observed (see Figure 1c). The LE-LC phase transition is shifted to 9.9 mN m-1, which is slightly higher than observed in the isotherm of DPPG/(KA)4K. The more hydrophobic side chains of α-amino butyric acid (Abu) of this peptide lead to a stabilization of the LE state, indicating that this uncharged residue is incorporated into the lipid monolayer hindering a regular arrangement of the lipid and the formation of LCACS Paragon Plus Environment

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domains. At higher surface pressure, the peptide is squeezed out and a pure lipid monolayer remains at the air-water interface. DPPG/(KV)4K The collected isotherms of mixtures composed of DPPG and (KV)4K (see Figure 1d) are different from the isotherms presented before. Increasing the peptide amount in the mixed monolayers shifts the area of the lift-off from 115 to 130 Å2 molecule-1. Similarly, the phase transition pressure increases from 9.4 mN m-1 for pure DPPG to 12.6 mN m-1 for a Rc = 1:1 (equivalent to a lipid to peptide ratio of 5:1) mixture of DPPG/(KV)4K. In addition, two plateau regions are visible at a charge ratio of Rc = 5:1 (equivalent to a lipid to peptide ratio of 25:1) when the charges of DPPG are not completely compensated by the peptide. The phase transition at lower surface pressure (Π = 10 mN m-1) is probably caused by the LELC phase transition of almost pure DPPG. The second phase transition occurring at Π = 12 mN m-1 could be due to a squeeze-out of the peptide with a concomitant condensation of the remaining lipid. For mixtures with higher peptide content, the double plateau in the compression isotherm vanishes and only the upper transition is observable at a fixed surface pressure of Π = 12.6 mN m-1. Again, peptide squeeze-out is complete at a surface pressure of

Π ~ 30 mN m-1. DPPG/(KL)4K Compression isotherms of mixed monolayers of DPPG with the most hydrophobic peptide (KL)4K (see Figure 1Error! Reference source not found.e) show a similar behavior as found for DPPG with bound (KV)4K. The values for the areas of lift-off are shifted to higher molecular area (115 to 125 Å2 molecule-1) compared to pure DPPG monolayers. The mixture with a charge ratio of Rc = 5:1 (equivalent to a lipid to peptide ratio of 25:1) shows again two phase transition plateaus, one at Π = 11 mN m-1, A = 85 Å2 molecule-1 and a second one at a higher surface pressure of 19 mN m-1, A = 60 Å2 molecule-1. ACS Paragon Plus Environment

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Apparently, due to its more hydrophobic properties (KL)4K is more stably bound to the airwater surface interacting with the PG molecules in the LE state so that a higher surface pressure is needed to squeeze-out the peptide into the subphase. For Rc = 1:1 only the transition at higher surface pressure remains. After complete squeeze-out of the peptide from the monolayer it is assumed that the peptide remains bound to the lipid monolayer due to the electrostatic attraction between both molecules. Especially for the system DPPG with (KL)4K with a charge ratio of 1:1 results from IRRAS experiments show that the peptide forms an antiparallel β-sheet structure underneath the lipid monolayer (A. Hädicke, A. Blume, unpublished results). The adsorption experiments also show in the LC-phase region that the PG monolayer become more condensed as the surface pressure decreases after peptide binding.35 This is also shown here from the difference in the isotherms with and without bound peptide (see section below and Figure 5). FT-IRRA-spectroscopy FT-IRRA-spectroscopy was used to determine the order of the acyl chains of DPPG as a function of surface pressure and peptide content. In particular, we were interested in the question of the origin of the double plateaus observed for certain DPPG/peptide mixtures. IRspectroscopy is suited for the determination of chain order, as the wavenumber of the asymmetric as well as symmetric CH2 vibrational band depends on the amount of gaucheconformers in the acyl chains.44-46 As the number of gauche conformers decreases drastically with the LE-LC phase transition, IRRAS is well suited to identify lipid phase transition plateaus in the isotherm and to discriminate them from plateaus of different origin, e.g. squeeze out. Figure 2 (top) shows the isotherm for a DPPG/(KL)4K mixture at Rc = 1:1 (equivalent to a lipid to peptide ratio of 5:1) together with the wavenumber of the antisymmetric and symmetric CH2 vibrational bands (νasCH2 and νsCH2, respectively). At this charge ratio only ACS Paragon Plus Environment

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one plateau is visible in the isotherms at elevated surface pressure (Π = 19 mN m-1). The spectroscopic data from IRRAS experiments show a condensation of the lipid component in the phase transition region (A = 75 to 50 Å2 molecule-1). The νasCH2 wavenumber decreases from 2923 cm-1 at a surface pressure of 9 mN m-1 in the LE state to 2919 cm-1 at surface pressures higher than 19 mN m-1 indicating the reduction of gauche conformers in the chains when the monolayer transforms into the LC state. The wavenumber of the νsCH2 band shows similar change. Thus in the region of the phase transition plateau not only the lipid chains become ordered, but also the peptide is squeezed out into the subphase as indicated by the value of the molecular area of the lipid shown in Figure 1e. Figure 2 (bottom) shows similar plots for other DPPG-peptide mixtures. The wavenumber of the νasCH2 band decreases in all cases with increasing surface pressure and indicating the LELC phase transition of the lipid. For the mixture DPPG/(KL)4K with Rc = 5:1 two plateaus appear in the isotherm (see Figure 1e). The wavenumber of the νasCH2 band first decreases steeply above a surface pressure of 10 mN m-1 (A = 80 to 65 Å2 molecule-1). Epifluorescence images of this region show that first domains of lipid in the liquid condensed state appear (see epifluorescence paragraph and Figure S2). Upon further compression a more gradual decrease of the wavenumber of the νasCH2 band up to Π = 40 mN m-1 is observed. The onset of this second, more gradual, decrease is concomitant with the onset of the second plateau in the compression isotherm, A < 60 Å2 molecule-1. This shows that lipid ordering also contributes to the second plateau of the isotherm. Similar isotherms for co-spread lipid-peptide monolayers showing 2 phase transitions were reported before.39,

47

However, in these cases, the peptides were longer and showed a

significant surface activity forming by themselves pure peptide monolayers at the air-water interface. (KL)4K is quite different compared to these peptides, as it shows essentially no surface activity on its own.35 For the longer peptides, the appearance of the second plateau at


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higher surface pressure was interpreted as being caused by a collapse of a peptide layer coexisting with the lipid monolayer at the interface. In our case, a stable peptide monolayer does not exist. At Rc = 1:1 a stable mixed LE state converts at higher surface pressure to a pure LC state monolayer containing only lipid, the peptide being squeezed-out into the subphase. At a charge ratio of Rc = 5:1, the system is apparently heterogeneous in the LE state with peptide-rich domains where charge compensation occurs due to bound peptide and peptide-poor domains. Compression of the monolayer first leads to a condensation of DPPG areas with low peptide content. In the epifluorescence images dark domains of condensed lipid are seen (for a detailed discussion see next section) surrounded by residual LE state lipids. The remaining peptide-rich DPPG domains with peptide bound with an assumed charge ratio of Rc = 1:1 is getting ordered at higher surface pressure (20 mN m-1) with a concomitant squeeze-out of the bound peptide into the subphase. The more gradual increase in ordered lipid domains is also visible in the epifluorescence images, as more and more of the surface is covered with condensed domains. In comparison, the IRRAS technique provides an average value of the whole surface leading to a gradual decrease of the wavenumber of the νasCH2 band.


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Figure 2: Top: Overlay of the compression isotherm with the wavenumber of the symmetric and antisymmetric CH2 stretching vibrational bands obtained by IRRA-spectroscopy for DPPG/(KL)4K Rc = 1:1. Bottom: Overlay of the compression isotherm with the wavenumber of the antisymmetric CH2 stretching vibrational band obtained by IRRA-spectroscopy for different mixtures of DPPG/peptide. Epifluorescence microscopy In our previous experiments, the peptides were injected into the subphase, and epifluorescence microscopy images were recorded during compression to determine the influence of the peptides on the morphology of the LC-domains.35 We performed now similar


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18 experiments with co-spread films of DPPG mixed with the different peptides. Our goal was to see whether differences in the preparation procedure would lead to different final states of the monolayer.

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Figure 3 shows the isotherms of DPPG/peptide mixtures, letters indicating the points at which epifluorescence microscopy images shown in Figure 4 were taken. The images of a pure DPPG monolayer in the LE state show a homogeneous brightly fluorescing film. When the DPPG monolayer is compressed, the first domains of lipids in the LC state occur at 7 mN m-1, slightly below the beginning of the phase transition region at ca. 9.4 mN m-1, A = 80 Å2 molecule-1 (see Figure 4a). The lipid dye Rh–DHPE used here prefers the LE state of a lipid monolayer so that LC-domains appear dark.35, 39 All the condensed domains have a propellerlike-shape with 2 or 3 arms (see Figure 4c) in agreement with previous observations.35 At the end of the plateau, the whole monolayer is covered with these regular condensed domains.


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Figure 4: Fluorescence microscopy images of pure and mixed DPPG peptide monolayers at the airwater interface (T = 20 °C, 100 mM NaCl) recorded at different surface pressure and charge ratios. Lipid dye: 0.01 mol% Rh–DHPE. The scale bar given in the image of pure DPPG monolayers applies to all other images shown in this figure. Figure 4 e-h shows images of a mixture of DPPG with (KV)4K at a charge ratio Rc = 1:1 (equivalent to a lipid to peptide ratio of 5:1). In this case no significant changes in domain shapes were observed compared to pure DPPG monolayers, the domains are only slightly smaller. These results are in agreement with the isotherm shown in Figure 3 which deviates only slightly from the isotherm of pure DPPG.


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The epifluorescence images for co-spread DPPG/(KL)4K films at a charge ratio Rc = 2:1 reveal the occurrence of the first condensed domains already at a surface pressure of 12.7 mN m-1, A = 75 Å2 molecule-1 (see Figure 4i), where the isotherm seems to indicate a film in the LE phase. Compression of the mixed film alters the shape of the condensed domains. The domains have more arms and the ends of them are sharper compared to LC-domains of pure DPPG (see Figure 4l). For a charge ratio Rc = 1:1, only one phase transition plateau was observed in the isotherm. Thus, the homogeneous LE state is stable up to 18.0 mN m-1, A = 80 Å2 molecule-1. At this surface pressure the first dark condensed domains are visible in the epifluorescence images (see Figure 4n). The shapes of the domains are completely different from the shapes observed for mixtures with lower peptide content. The first domains formed upon compression act as nuclei and consecutively small filaments grow from the nucleation seeds in different directions (see Figure 4o). An explanation for such elongated domains is the occurrence of a reduced line tension between the LE and LC state due to the bound peptide still present in the surrounding LE state. Preliminary results obtained with a fluorescently labeled peptide of similar structure ((KGG)4K) showed that this peptide partitions preferentially at the phase boundaries between LE and LC domains (Hädicke and Blume, unpublished results). We therefore think that this is also the case for the peptides used here. The filaments are curled which might be a result of the tilted acyl chains of the lipid in the LC state. Similar shapes of protruding curls along the boundaries of condensed domains can be seen for DPPC/POPG/cholesterol mixtures as cholesterol also lowers the line tension between the two phases.48 Also in lipid monolayers with incorporated polymers carrying a cholesterol or perfluorinated alkyl anchor, the line tension between LE and LC-domains is reduced leading to domains with “fuzzy” shapes.25, 49


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Comparison with adsorption experiments We now want to discuss similarities or differences in monolayer behavior between the adsorption experiments and co-spreading experiments. Figure 5 shows the difference in surface pressure ∆Π observed in the isotherms for pure DPPG monolayers and those mixed with peptides. The mixtures were prepared either by co-spreading of lipids and peptide (full lines) or by peptide adsorption from the subphase to a monolayer with different initial surface pressure values (symbols). When comparing these curves one has to be aware that in the cospreading experiment the ratio of lipid to peptide is given by the composition of the organic solution which is spread onto the water surface. In the adsorption experiments, the total concentration of the peptide in the subphase was fixed to 3 µM. In adsorption experiments, the peptide was always in excess (charge ratio Rc 0.1-0.3) to assure that a saturation of the lipid monolayer with bound peptide was occurring.35 In the co-spread monolayers the charge ratio was Rc = 1:1 and the peptide concentration was therefore lower than in the adsorption experiment. A rough calculation of the maximal concentration in the subphase for a mixture with Rc = 1:1 if all peptide would partition into the subphase yields a concentration of ~0.5

µM. This concentration is just the saturation concentration as shown before in the adsorption experiments. We can therefore conclude that almost all of the peptide remains bound to the monolayer after spreading. Figure 5 shows that ∆Π is positive at low surface pressure (LE state DPPG) as the peptide can be incorporated into the film increasing the surface pressure. With increasing film pressure,

∆Π first increases and then goes to zero or even becomes negative. In the adsorption experiments at high initial surface pressure of the preformed film, when the DPPG monolayer is in its LC state, more negative ∆Π values are observed, indicating electrostatic binding with charge compensation only, leading to a condensation of the film. This effect is not as pronounced in the co-spread monolayers. One reason could be that upon compression of the


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22 mixed film some peptide still remains at the interface even at higher surface pressure due to slow desorption kinetics. In this case a decrease in surface pressure due to charge screening can be compensated by residual incorporated peptide residing in the expanded areas of the film. Another reason is the lower peptide concentration present in the co-spread films leading to a reduced condensation of the LC state. Thus, the observed slight differences between cospread monolayers and those obtained after peptide adsorption can be easily explained by differences in charge ratio and by different kinetics of adsorption and desorption.

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Differences in surface pressure ∆Π observed between monolayers of pure DPPG and DPPGpeptide monolayers as a function of surface pressure Π. The ∆Π data for the mixed peptidelipid monolayers were either obtained from co-spread monolayers (solid lines) or from adsorption experiments with preformed monolayers at different surface pressures Π (symbols). Data from peptide adsorption experiments (symbols) were taken from Hädicke & Blume, 2016.35

Co-spreading of (KL)4K with other phospholipids (KL)4K/DPPC and (KL)4K/DMPE DPPC and DMPE are zwitterionic lipids. These two lipids were chosen as they both show similar isotherms at room temperature. In our previous investigations on the adsorption of (KL)4K to these lipid monolayers, no changes in surface pressure were observed.36 When the peptide is co-spread with the lipids onto the surface, the initial situation is different. After ACS Paragon Plus Environment

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23 solvent evaporation the peptide might remain at the air-water interface interacting with the lipids. However, the isotherms of mixtures of the peptide (KL)4K with the zwitterionic lipids DPPC and DMPE show almost no changes in the lift-off area compared to the pure lipids (A = 90 and 85 Å2 molecule-1, respectively) (see Figure 6). As both lipids are zwitterionic, the interaction of the peptide with the lipids must be driven solely by hydrophobic interactions. These are apparently too weak to keep the peptide bound to the interface. Instead, the peptide is desorbed from the surface into the subphase after spreading from the organic solution and an almost pure lipid film remains at the air-water interface.

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Figure 6: Π-A isotherms of co-spread mixtures of zwitterionic lipids with (KL)4K peptide: a) DMPE/(KL)4K, b) DPPC/(KL)4K. For comparison, the isotherms of pure lipids on 100 mM NaCl solution as subphase are also shown. (KL)4K co-spread with different phosphatidylglycerols (KL)4K/POPG Pure POPG forms only LE state monolayers due to one unsaturated oleoyl chain. The LE monolayer is stable up to a surface pressure of 30 mN m-1 only. Further compression leads to film collapse. For a mixed monolayer with a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1) the area per molecule is increased, shifting the lift-off to higher area values (from 110 to 125 Å2 molecule-1) (see Figure 7a). The increase in area in the LE state is similar to the increase ACS Paragon Plus Environment

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24 observed for a mixture with DPPG. A squeeze out of the peptide was not observed up to the film collapse at a surface pressure of Π > 40 mN m-1. Thus, the peptide seems to be incorporated into the LE state of the POPG monolayer stabilizing the liquid-expanded state.

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Π-A isotherms of co-spread mixtures of phosphatidylglycerols with (KL)4K peptide of different charge ratios: a) POPG/(KL)4K, b) DMPG/(KL)4K, c) DSPG/(KL)4K. For comparison, the isotherms of pure lipids on 100 mM NaCl are also shown. (KL)4K/DMPG For DMPG with shorter acyl chains than DPPG, the LE-LC phase transition region occurs at a surface pressure of 40 mN m-1. For mixtures with low amounts of peptide, the isotherms are more or less unchanged, only the transition pressure for the onset of the LE-LC phase transition is slightly increased due to the incorporation of the peptide (see Figure 7b). At a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1), the effect of the peptide on ACS Paragon Plus Environment

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the DMPG monolayer is similar to the effects observed with POPG monolayers in the LE state. The lift-off is shifted to a higher area (from 115 to 135 Å2 molecule-1) but the shape of the isotherm is more or less unchanged. The onset of the LE-LC phase transition is slightly shifted to higher surface pressure. The peptide seems to be squeezed out of the film when it enters the LC state. However, this is difficult to determine as film collapse occurs concomitantly. (KL)4K/DSPG Elongation of the saturated acyl chain of PGs, as in DSPG, results in a direct transition from the gas-analogue state to the LC state upon compression.50 The interaction of the peptide with DPSG in the LE state leads to the appearance of a plateau region at a surface pressure of 6 mN m-1, indicating an LE-LC phase transition. Thus at 20 °C the phase sequence gas-analogue state => LE => LC appears. For pure DSPG the lift-off area for the gas-analogue-LC transition is observed at 53 Å2 molecule-1. For the mixed film with a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1) the lift-off area for the LE-LC transition is ~ 105 Å2 molecule-1 (see Figure 7c) and thus only slightly lower than the lift-off area observed for mixed films with DPPG (see Figure 1e). The appearance of an LE-LC phase transition is a hint for an incorporation of the peptide into the lipid acyl chain region of the LE state and its stabilization. Further compression of the monolayer into the LC state forces the peptide into the aqueous subphase as observed before and the monolayer finally consists again only of lipid molecules with surface bound peptides. It is assumed that the peptide remains bound to the lipid monolayer as discussed before in the system with DPPG. Epifluorescence microscopy Figure 8 shows images of pure films of DMPG and DSPG with and without bound (KL)4K at a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1). Images of monolayers of DPPG have been shown before in Figure 4. In pure DPPG monolayers the LC-domains have


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the familiar propeller-like shape, in monolayers of DMPG, the condensation to the LC state occurs at much higher surface pressure. The domains are smaller and more rounded (Figure 8c). A fully condensed monolayer cannot be reached because of film collapse. In pure DSPG monolayers at almost zero surface pressure, a coexistence between gas-analogue and LC state is present. The amount of LC state increases upon compression. The images show brighter network-like areas against a black background. As the fluorescent dye is usually excluded from the condensed state, these brighter areas must arise from the gas-analogue state lipids containing the dye. However, the dye is probably aggregated in these areas and is not homogeneously distributed. Addition of (KL)4K at a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1) changes the domain shapes. For DMPG monolayers the LC-domains are much smaller and not round any more. With increasing surface pressure filaments grow from their edges (see Figure 8e-g). These domain shapes indicate a reduction in line tension between LE and LC state, meaning that the (KL)4K content is increased at the LC-domain boundaries. This is similar to what was observed before for DPPG monolayers. For DSPG monolayers with bound (KL)4K the images change dramatically (see Figure 8n-p). Now, a bright background is seen at very low surface pressure with black LC-domains, which increase in area with increasing surface pressure. The LC-domains of DSPG themselves seem to cluster. A reason for such behavior might be the increased hydrophobic interaction among the longer acyl chains and the fact that the acyl chains are not tilted with respect to the monolayer normal in the domains, as seen for DPPG.This clearly indicates that the plateau region observed in the isotherm (see Figure 7c) is indeed due to a transition from the LE to the LC state, the LE state containing the dye. This agrees with the upshift of the plateau region found for the other PG monolayers with bound (KL)4K (see Figure 7). The LC-domains formed in DSPG monolayers are not rounded but have irregular star-like shapes, indicating


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again that the peptide is line active. Besides the line activity of the peptide, the acyl chain tilt also influences the shape of the domains. For DSPG having no chain tilt the protrusions of the domains are straight.51-52

Figure 8: Fluorescence microscopy images of pure DMPG or DSPG monolayers at the air-water interface (T = 20 °C, 100 mM NaCl) and their mixtures with (KL)4K of a charge ratio of 1:1 recorded at different surface pressures. Lipid dye: 0.01 mol% Rh–DHPE. The scale bar in the image for DMPG monolayers applies to all other images in this figure.


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Mixed films of (KL)4K with other anionic phospholipids with different headgroups (KL)4K/DMPS DMPS has a slightly larger headgroup than DMPG, but due to less bound water molecules its effective size is smaller.53-54 Thus, the surface pressure for the LE-LC phase transition is reduced and occurs at 18 mN m-1 compared to 40 mN m-1for DMPG. The headgroup is singly charged at pH 7 on 0.1 M NaCl solution.55 Co-spreading of DMPS/(KL)4K mixtures with low peptide content creates only minor changes in the isotherms (see Figure 9a). For a 1:1 charge ratio (equivalent to a lipid to peptide ratio of 5:1), the phase transition pressure is increased from 18.5 mN m-1 to 28.6 mN m-1, and the lift-off area is shifted from 95 Å2 molecule-1 up to 120 Å2 molecule-1. Thus, the peptide occupies space at the air-water interface when the lipid monolayer is in the LE state. Upon compression into the LC state the peptide is squeezed out into the subphase, as the isotherms overlap at high surface pressure. (KL)4K/DMPA The interaction of (KL)4K with DMPA (see Figure 9b) shows similar features as already reported for mixtures with DPPG. At pH 7 on 0.1 M NaCl as subphase, DMPA is singly charged.55-57 The value for the lift-off area is shifted to higher areas (from 85 to 95 Å2 molecule-1). For an intermediate charge ratio of 5:1 (equivalent to a lipid to peptide ratio of 25:1), a double plateau behaviour is observed as discussed before for DPPG monolayers. For a mixture with the charge ratio of 1:1 the LE-LC phase transition pressure is shifted by 10 mN m-1 up to 16.5 mN m-1compared to pure DMPA monolayers. (KL)4K/TMCL Mixed monolayers of the lipid TMCL with (KL)4K (see Figure 9c) show similar changes in the isotherms as seen above for monolayers of DPPG and DMPA with bound peptide. TMCL


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29 has four acyl chains and the headgroup has two ionizable phosphate diester groups. There has been a considerable debate in the past over the pKa-values of the two phosphate groups. It was suggested that the second pKa of TMCL is much higher than expected (> 7.0) so that at pH 7 TMCL would not be completely doubly charged. However, more recent experiments using IR and NMR and also our own experiments using IR (Finger and Blume, unpublished) showed that TMCL carries indeed two negative charges at pH 7.58-60 Therefore, only the charge ratio is the relevant parameter for a comparison with other anionic lipids. The molecular area at lift-off is approximately twice as high compared to the value for singly charged lipids with two chains. a

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Π-A isotherms of co-spread mixtures of different anionic phospholipids with (KL)4K at different charge ratios: a) DMPS/(KL)4K, b) DMPA/(KL)4K, c) TMCL/(KL)4K. For comparison, the isotherms of pure lipids on 100 mM NaCl are also shown. d) Surface pressure change ∆Πtrans of the observed phase transition as a function of 1/Rc at the compressibility maximum. ACS Paragon Plus Environment

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For TMCL mixtures with the peptide, a double plateau is again observed in the isotherm for a charge ratio of 5:1 (equivalent to a lipid to peptide ratio of 12.5:1). At a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 2.5:1), only a single plateau is seen and the transition pressure is higher than for the pure TMCL monolayer (see Figure 9d). The shift in transition pressure is slightly lower with 7 mN m-1 compared to the monolayers containing DPPG, DMPA or DMPS where at a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1), the phase transition occurs at a surface pressure of 9.4 – 10.1 mN m-1 above the phase transition of the pure lipids. Epifluorescence microscopy Pure DMPA (Figure 10a-d) and TMCL films (Figure 10i-m) show large rounded or Pac-Manshaped LC-domains upon compression, indicating that the line tension between the two phases is high. The domain shapes change strongly after addition of (KL)4K, as domains with long curved filaments are formed which are much smaller in size. The change in line tension leading to the increase in line boundaries is much larger compared to the effects observed for PGs. As DMPA and TMCL are not tilted in the LC state61 the domains are not chiral as in the case of DPPG but show filamentous extensions in all directions leading to a fuzzy structure. In contrast, fractal flower-like domains are seen in pure DMPS monolayers which are very large as observed before in DMPA and TMCL monolayers. With bound (KL)4K the LCdomains become round and equal in size and are regularly packed (see Figure 10y). The occurrence of these hexagonal patterns results from an equilibrium between electrostatic repulsion and line tension as discussed before62-63 and observed with DPPG monolayers with other (KX)4K peptides which are less hydrophobic, for instance, with bound (KA)4K.35 Epifluorescence pictures and the corresponding compression isotherms of the 5:1 mixtures are now shown in the SI (see Figure S2 and S3). There are no new features visible as seen before for the 2:1 mixtures.


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Figure 10: Fluorescence microscopy images of pure DMPA, TMCL or DMPS monolayers at the airwater interface (T = 20 °C, 100 mM NaCl) and their mixtures with (KL)4K of a charge ratio of 1:1 recorded at different surface pressures.. Lipid dye: 0.01 mol% Rh–DHPE. The scale bar in the image for DMPA monolayers applies to all other images in this figure.


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The results of the epifluorescence experiments show again that the nature of the phospholipid headgroup of the anionic phospholipids has subtle influences on the binding properties of the peptides. We have shown before for lipid bilayer vesicles that the size of the lipid headgroup affects the binding properties and that tigher binding is possible when the headgroup is smaller as in PAs and CLs. A lower binding strength is observed for lipids with larger headgroups, such as PS.38 Data from IR measurements show that the peptide (KL)4K obtains a β–sheet structure when bound to gel-phase bilayers37 or LC state monolayers of DPPG.64 The formation of a β–sheet structure is more likely when the binding is increased as for DMPA and TMCL monolayers where the headgroups have a smaller size. Therefore, the extreme reduction in line tension observed for these two lipids might be caused by an increased binding and increased formation of β–sheets at the interface between the different domains. Summary and Conclusions When cationic peptides with the structure (KX)4K are co-spread with anionic lipids from organic solvent, changes in the shape of the monolayer isotherms are seen compared to those of pure lipid monolayers. The peptide sequence has a defined effect on the shape of the isotherms of the lipid-peptide films at the air-water interface. A broadened phase transition region is observed for co-spreading DPPG with the more hydrophilic peptides (KG)4K, (KA)4K and (KAbu)4K. For a charge ratio of 1:1 (equivalent to a lipid to peptide ratio of 5:1), a shift of the phase transition pressure to higher values is detected. Thus, destabilization of LC state upon peptide incorporation into the acyl chain region of the lipids in the LE state is occurring, leading to an increase in transition pressure. This is equivalent to a decrease in transition temperature at constant surface pressure. An incorporation of the more hydrophobic peptides (KV)4K and (KL)4K into the LE state of the monolayer shifts the isotherms to larger molecular area, i.e. the lift-off area is increased. For most lipids co-spread with (KL)4K, an area increase of about 10 - 20 Å2 molecule-1 is observed for lipids in the LE state. From ACS Paragon Plus Environment

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molecular modelling the length and thickness of (KL)4K in its extended conformation can be estimated to be 30 and 5 Å, respectively. Thus, the molecular area of the peptide is ~ 150 Å2 when the side chains are all pointing into the air. For a charge ratio of 1:1 one would expect an increase of molecular area by 30 Å2 as a maximum. As the lysine side chains are probably intercalated between the lipid headgroups, a smaller molecular area increase has to be expected. The observed area increase thus indicates that almost all of the peptide remains in the film when it is in the LE state. The LE-LC phase transition pressure is increased to a greater extent when (KL)4K is bound than for the less hydrophobic peptides. At a charge ratio of 5:1 (equivalent to a lipid to peptide ratio of 25:1), isotherms with two plateau regions were recorded. The first phase transition corresponds to the formation of condensed domains as observed by epifluorescence microscopy and IRRA-spectroscopy, whereas in the second phase transition region, a reorganization of the peptide is occurring, i.e. the peptides are squeezed-out into the subphase. The system is heterogeneous in the following sense. At low surface pressure LC-domains of pure PG without bound peptide are formed, whereas areas with bound peptide with a charge ratio 1:1 need a higher surface pressure to be condensed into the LC state with a concomitant peptide squeeze-out. When the total amount of the added peptide is increased to a charge ratio of 1:1, both phase transitions occur at the same surface pressure. In this case, condensation to the LC state is concomitant with a squeeze-out of the peptide into the subphase. Isotherms obtained after injecting the peptide into the subphase and those obtained from co-spreading are similar. The differences can be explained by different peptide concentrations in the two experiments and by the fact that the kinetics of adsorption and desorption of the peptides can play a role. Epifluorescence images of films of DPPG/(KL)4K mixtures show first nucleation sites for LCdomains from which thin filaments grow when the film is further compressed. Similar shapes


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of the condensed domains are observed for DMPG, a shorter chain analogue of DPPG. The increase of the required area for the lift-off into the LE state was also observed for POPG and DSPG monolayer. In the case of pure DSPG monolayers, the transition from the gas-analogue to the LC state leads to aggregation of the fluorescent dye in the gas-analogue state. Addition of (KL)4K at a charge ratio of 1:1 increases the transition pressure so that the transition occurs now from the LE to the LC state at room temperature. Star-shaped LC-domains are observed in this case. Mixing (KL)4K with negatively charged lipids having other headgroups shows the importance of the chemical structure of the lipid headgroup and its size on the strength of the interactions. The LE-LC phase transition at a charge ratio of 1:1 is increased by 6 - 10 mN m-1, dependent on the structure of the anionic lipid headgroup. A reduction of the line tension between the LC-domains of the pure lipids and the surrounding LE state of the mixed lipid-peptide monolayer creates domains with fractal structure and with long filaments. An exception from this behavior is the mixture DMPS having a larger headgroup with (KL)4K. In this case a regular lattice of round domains appears which is stabilized by repulsive electrostatic interactions between LC-domains having a higher dipole density than the surrounding LEphase. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (GRK 1026 Conformational Transitions in Macromolecular Interactions, Project A1). Supporting Information Graphs of compressibility vs. molecular area for lipid-peptide mixtures, epifluorescence images for lipid-peptide mixed monolayers at a charge ratio of Rc = 5:1, and monolayer isotherms of (KL)4K-lipid mixtures with different anionic phospholipids with indications where epifluorescence images were taken.


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