CHAPTER INTERACTION BETWEEN ANTIMICROBIAL PEPTIDES AND LIPID MEMBRANE LAYERS 7.1 Introduction V4 was shown to first bind to the membranes and finally induce membrane permeation by membrane aggregation and disruption. However, the intermediate process is not clear. In this chapter, monolayer is mainly used to investigate the process of V4 inserting into membranes and the comparision of V4 to other antimicrobial peptides is also shown. 7.2 Materials and methods Materials POPG, POPC and DPPG were purchased from Avanti. Solvent chloroform (HPLC grade) and methanol (HPLC grade) and antimicrobial peptide magainin (M2), melittin (ME) and polymyxin B (PB) were purchased from Sigma-Aldrich. V4 peptide was synthesized by Genemed. The purity of different peptides has been presented in previous chapters. All the materials were used without further purification. Instrumentation Langmuir film balance (model 601M) (NIMA Technology Ltd. England) was used for experiments. The instrument includes a 105 cm2 trough connected with an external circulator for temperature control, two mechanically coupled barriers, a surface pressure sensor using Wihelmy plate, a sapphire window and a dipper well (25mm stroke). An interface unit (IU4) connected the film balance and the computer. An operating software 141 (version 5.16) provided by NIMA Technology Ltd was used to collect data. The cleanness of the trough was checked by closing and opening of the barriers to ensure that surface pressure did not vary by more than ±0.1 mN/m. Monolayer isotherms POPG and POPC were dissolved in chloroform with a concentration of 0.2 mM. Due to the low solubility of V4 in chloroform, V4 was first dissolved in the minimal volume of methanol to prepare a clear solution. Additional chloroform was then added to prepare a solution with V4 concentration of 0.2mM. Required volume of POPG or POPC was mixed with V4 to form lipid/V4 mixture with V4 percentage of 0%, 5%, 10%, 20%, 33%, 50% and 100%. A syringe was cleaned completely and an appropriate volume of individual lipid/V4 mixed solution was drawn and carefully deposited on the water surface in a drop-wise manner, making sure that the surface pressure did not change after deposition. The monolayer of lipid in the absence or in the presence of V4 formed spontaneously on the air-water interface. After solvent evaporation for 10 minutes, the monolayer was compressed with a rate of cm2/min and the isotherm curve was record. Each curve was repeated at least twice for reproducibility. Penetration studies POPG and POPC were dissolved in chloroform and DPPG was dissolved in the mixture of chloroform and methanol (v/v=3:1) with a final lipid concentration of 0.2 mM. All the studied antimicrobial peptides were dissolved in water with high concentrations as stock solution. Required volume of lipid solution (usually 60 µl) was drawn by using a clean 142 syringe and spread on the water surface carefully in a drop-wise manner, making sure that the surface pressure did not change after lipid deposition. The lipid monolayer formed spontaneously on the air-water interface. After solvent evaporation for 10 minutes, the monolayer was compressed with a rate of cm2/min to a target surface pressure. The lipid monolayer was allowed to adjust until a constant molecular area was achieved. Afterward an appropriate volume of peptide solution was injected underneath the monolayer into the subphase, generating different peptide concentration in the trough. The surface pressure change with time with fixed molecular area was record. All the experiments were done at 37 °C and each penetration experiment was repeated at least twice for reproducibility. A water subphase but not buffer was used to avoid crystallization of salt on the sample which would interfere with the imaging of sample in the AFM experiments. AFM experiment A monolayer which was penetrated by antimicrobial peptides was transferred to freshly cleaved mica (Electron Microscopy Sciences, USA) by vertically placing the mica in the water subphase before lipid was spread on the water surface. After the penetration experiment, the surface pressure was kept constant at the surface pressure of complete penetration. The mica was slowly extracted from the subphase to the air phase with a constant rate of mm/min. A lipid monolayer was obtained by compressing the lipid monolayer to a certain surface pressure and extracted from the mica from the subphase at the constant target surface pressure. The monolayer with or without peptide on the mica was dried in a desiccator overnight before AFM imaging. 143 AFM experiment was performed in air on the NanoScope IIIa MultiMode Scanning Probe Microscope manufactured by Digital Instruments Inc. (Santa Barbara, CA 93117, USA). Topographic images were acquired in tapping mode. The typical scan rates ranged from to 1.25 Hz depending on the scan size. The monolithic silicon probes (NanoWorld AG, Switzerland) with a cantilever length of 125 µm and force constant of 42 N/m were used for measurements. Images was obtained and analyzed by the Nanoscope software provided by the company. Images from at least two different sample prepared on different days with several macroscopically separated areas on each sample were acquired for data reproducibility. Representative images are shown. Insertion study of V4 into POPG bilayer Dual polarization interferometry was used to study the insertion of V4 into solid supported bilayers. This technique allows the opto-geometrical properties (density and thickness) of adsorbed layers at a solid-liquid interface to be determined 132 . When a peptide is introduced into the thin lipid bilayer, the changes of average thickness and average density (through the refractive index) of the lipid bilayer as well as the mass can yield information of how the peptide interacts with the lipid bilayer. Experiments were done on an AnaLight ® Bio 200 system. POPG SUVs were prepared and deposited on an amine modified surface sensor chip. After obtaining a stable POPG bilayer on the surface, V4 was injected and the density and mass change of the POPG bilayer were recorded. 144 7.3 Results and discussion 7.3.1 Isotherm studies of V4 interacting with POPG and POPC 7.3.1.1 Isotherms of lipid monolayers The surface pressure (π) and molecular area (A) isotherms for POPG and POPC monolayer at the air-water interface at 37 °C are shown in Fig. 7.1. When the lipid monolayer was compressed, the isotherm for POPG and POPC began to rise at a molecular area of 107 Å and 96 Å, respectively. With increasing compression, the surface pressure increased continuously until the collapse pressure of 45.6 mN/m and 44.5 mN/m for POPG and POPC, respectively. The shape of isotherms of POPG and POPC monolayer was similar especially at low molecular area, which indicated that the packing of POPG and POPC lipid molecules was similar. POPG and POPC are both unsaturated lipids containing one double bond. They have the same hydrophobic alkyl chains and differ in the headgroup. The molecular packing of the monolayer is mainly dependent on the hydrophobic interaction between the alkyl chains of the lipid molecules. The same alkyl chain of POPG and POPC allowed similar molecular packing so that POPG and POPC showed similar isotherms. Thus hydrophobic interaction played a significant role during the compression. Although POPG bears a negative charge, which might impose a repelling effect between POPG molecules especially when the molecules were close, there was not much difference between the POPG and POPC isotherms, indicative of the negligible effect of electrostatic interaction. 145 Fig. 7.1 Isotherms of POPG and POPC monolayers 7.3.1.2 Isotherms of mixed lipid/V4 monolayers Fig. 7.2 shows the surface pressure (π) and molecular area (A) isotherms of POPG/V4 and POPC/V4 monolayers at the air-water interface at 37 °C. The pure V4 showed strong surface activity compared with lipid. At a molecular area of 125 Å, the isotherm of V4 began to rise. With increasing compression, the surface pressure of V4 monolayer continuously increased to 45.0 mN/m, which was comparable to the collapse pressure of POPG and POPC. When V4 was incorporated into the lipids, the isotherms of the mixed POPG/V4 and POPC/V4 monolayer shifted right to the high molecular areas with increasing percentage of V4. The collapse pressure for all isotherms was similar except for the POPG/V4 mixture with 50% V4 incorporation, which was an incomplete isotherm because the two barriers were too close. The shape change of isotherms was complicated. The isotherms of POPG/V4 with V4 percentage of 5% and 10% were similar to the isotherm of pure POPG. When V4 percentage increased to 20% or higher, there was a kink at surface pressure of 30 mN/m, which indicated that V4 peptide might induce a 146 phase transition. Above 30 mN/m, the increase of surface pressure due to the compression slowed down. The presence of V4 in the POPC monolayer with V4 percentage of 5% did not induce much change in the isotherm. However when the percentage of V4 increased to 10% and higher, the shape of the mixed POPC/V4 isotherms was similar to pure V4 isotherm. Comparing all the molecular areas at which the surface pressure began to increase (lift-off area), it was found that with increasing incorporation of V4, the lift-off areas gradually increased for both lipids, which indicated that V4 had an area-expanding effect on the lipid monolayer at low surface pressure. 147 Fig. 7.2 Isotherms of mixed POPG/V4 and POPC/V4 monolayers 7.3.1.3 Miscibility analysis of monolayers When the studied components were mixed to form monolayers on the air-water interface, it was not easy to determine if the components were really miscible or not from the direct measurement. Analysis of the monolayer isotherm may provide useful information to 148 determine the miscibility of the studied components in the monolayer. Each pure component has its own collapse pressure. In a two-component system, if the components were immiscible, two collapse pressures would be observed at the corresponding collapse pressure of pure components. However if the two components were miscible, only one collapse pressure would be obtained220. Fig. 7.2 showed that all mixed monolayers had one collapse pressure for both POPG and POPC, which gave an indication that V4 was miscible with POPG and POPC. According to the phase rule of Defay and Crisp221, in a two-component system, if the components are completely miscible for all the ratios, there is only one degree of freedom at constant temperature and pressure, assuming no externally imposed electrical potentials. Therefore when the percentage of V4 in the mixed lipid/V4 solution varies, the surface pressure will vary correspondingly at fixed molecular area. If V4 and lipid mix ideally, the ideal surface pressure can be calculated by π A,ideal = X (π ) A + X (π ) A (7.1) π1 and π2 are the surface pressure of the pure lipid and V4 at a given molecular area A, respectively. X1 and X2 imply the percentage of lipid and V4, respectively. If the mixing is ideal, the surface pressure will vary linearly with the percentage of V4 in the mixed monolayer. However if the mixing is non-ideal, a deviation from the linear relationship will be obtained. From the excess surface pressure, which describes the deviation from the ideal mixing, interaction between V4 and lipid could be deduced. The excess surface pressure πA,ex can be obtained by the difference between the surface pressure of the real mixed monolayer πA, exp and the ideal mixed monolayer according to Eq. 7.2. 149 π A,ex = π A,exp − π A,ideal (7.2) Fig. 7.3 shows that the surface pressure varied with different percentage of V4 incorporating into POPG and POPC monolayer at different molecular areas. An apparent difference between the different molecular areas was observed for both lipids. At high molecular area, which the molecules were loosely packed, the surface pressure increased gradually with the increasing percentage of V4. When there was 50% V4 incorporated into the lipid monolayer maximal surface pressure was obtained. With increasing molecular area, the surface pressure difference between the different molecular areas became smaller. At low molecular area, the curve became flat (POPG) or the maximal surface pressure shifted to low percentage of V4 incorporation (POPC). Fig.7.3 Surface pressure of lipid monolayers incorporated with different percentage of V4. Left: POPG; Right: POPC. The excess surface pressure, which indicates the deviation from ideal mixing is shown in Fig. 7.4. For the POPG lipid, except at a low molecular area of 50 Å, the surface pressure at the other molecular areas all showed positive deviation from linearity. The positive excess surface pressure indicated that V4 had a surface pressure increasing effect on the monolayer, which was equivalent to an area expanding effect. 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Chem. 14, 6065-6074 (2006). 209 [...]... confirmed the role of electrostatic interaction in the penetration process 169 7 .3. 3 AFM studies of antimicrobial peptides interacting with lipid monolayers 7 .3. 3.1 AFM images of pure lipid monolayers The morphology of pure POPG, POPC and DPPG monolayers as references is shown in Fig 7. 13 In the absence of antimicrobial peptides, the AFM image of the pure lipid monolayer displayed a regular and flat... area-expanding effect on both lipid monolayers One of the possible reasons might be the conformational change due to the interaction between V4 and lipid When there was no V4, the alkyl chains of the lipid molecules extended upward to the air phase and the headgroup contacted with water phase The hydrophobic interaction between the hydrophobic part of V4 and alkyl chains of the lipid made the alkyl... penetrate but they were not capable of enwrappping the monolayer Therefore monolayers cannot be disrupted by the peptides 167 7 .3. 2.4 Effect of lipid packing on the penetration of V4 The pressure of vesicles is about 30 mN/m198-201 In order to compare the penetration of V4 into vesicle and monolayer and mimic live cell pressure, the target pressure of 30 mN/m was chosen to investigate the penetration of V4... before, the determining factor for the isotherm is the hydrophobic interaction between the alkyl chains of lipids and charge nearly has no effect Compared with some saturated lipids, POPG and POPC both have high lift-off molecular areas at which the surface pressure began to rise from zero upon compression2 23 Because of the unsaturated structure, the kink in the alkyl chains, which is formed by the double... Electrostatic interaction leads to the absorption of higher amount of peptide on the headgroup of POPG, which increased the opportunity of peptides penetrating into the lipid monolayer Once the peptide molecules absorbed on the monolayer, the hydrophobic interaction drove the peptide molecules to penetrate into the monolayer and induced an increase in surface pressure However, due to the lack of electrostatic interactions... that determined the binding Therefore the binding of V4 to mixed lipid SUVs and pure POPG SUVs was similar However in the monolayer experiments, the absorption of the peptide molecules was dependent on the charge amount of the lipid monolayer The more charge the monolayer harbored, the more V4 molecules absorbed on the monolayer, which led to a higher penetration Therefore the amount of charge exerted... interacting with lipid monolayers 7 .3. 2.1 Penetration of antimicrobial peptides into POPG monolayers The presence of antimicrobial peptide in the subphase induces a surface pressure change, which indicates the penetration or insertion of the peptide into the lipid monolayer The interaction between magainin 2, melittin, polymyxin B and V4 with POPG monolayers is shown in Fig 7.7 Before peptide was injected, the. .. all studied lipids, which indicated that these lipid molecules formed a homogenous monolayer organization The section analysis of the pure lipid monolayer as shown in Fig 7.14 provided the cross-section profile of monolayer with regard to height difference It can be observed that the height difference of the pure lipid monolayer was very small The height difference of the pure POPG, POPC and DPPG monolayer... illustrated in the figure by the two arrows was 0.291, 0.226 and 0 .37 7 nm, respectively, which confirmed the flatness of the pure lipid monolayers Fig 7. 13 AFM topographic images of pure POPG, POPC and DPPG monolayers 170 Fig 7.14 Section analysis of pure POPG, POPC and DPPG monolayers 7 .3. 3.2 AFM images of POPG monolayers penetrated by antimicrobial peptides Fig 7.15 shows the AFM images of POPG monolayers... Fig 7.11 Comparison of V4 penetrating into different lipid monolayers 7 .3. 2 .3 Penetration of V4 into different lipid monolayers The interaction of V4 with different lipid monolayers was investigated to examine the penetration ability of V4 into different membranes Fig 7.11 illustrates the comparison of V4 penetrating into POPG, POPC, DPPG and a mixed monolayer of POPG/POPE (1/2) With increasing peptide . 145 7 .3 Results and discussion 7 .3. 1 Isotherm studies of V4 interacting with POPG and POPC 7 .3. 1.1 Isotherms of lipid monolayers The surface pressure ( π ) and molecular area (A) isotherms. between the POPG and POPC isotherms, indicative of the negligible effect of electrostatic interaction. 146 Fig. 7.1 Isotherms of POPG and POPC monolayers 7 .3. 1.2 Isotherms of mixed lipid/ V4. for the isotherm is the hydrophobic interaction between the alkyl chains of lipids and charge nearly has no effect. Compared with some saturated lipids, POPG and POPC both have high lift-off