Investigation of the interaction of antimicrobial peptides with lipids and lipid membranes 3

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. The more V4 was incorporated, the greater the effect was until a maximal value was reached with V4 incorporation of 33% to 50% dependent on different molecular areas. At the molecular 150 27. Kaufmann, S. H. & Earnshaw, W. C. Induction of Apoptosis by Cancer Chemotherapy. Exp. Cell Res. 256, 42-49 (2000). 28. Winder, D., Gunzburg, W. H., Erfle, V. & Salmons, B. Expression of Antimicrobial Peptides Has an Antitumour Effect in Human Cells. Biochem. Biophys. Res. Co. 242, 608-612 (1998). 29. Chernysh, S. et al. Antiviral and antitumor peptides from insects. P. Natl. Acad. Sci. U. S. A. 99, 12628-12632 (2002). 30. Raj, P. A. & Dentino, A. R. Current status of defensins and their role in innate and adaptive immunity. FEMS Microbiol. Lett. 206, 9-18 (2002). 31. Mackewicz, C. E. et al. alpha-Defensins can have anti-HIV activity but are not CD8 cell anti-HIV factors. AIDS 17, F23-F32 (2003). 32. Chang, T. L., Vargas, J., Jr., DelPortillo, A. & Klotman, M. E. Dual role of alphadefensin-1 in anti-HIV-1 innate immunity. J. Clin. Invest 115, 765-773 (2005). 33. Bourinbaiar, A. S. & Coleman, C. F. The effect of gramicidin, a topical contraceptive and antimicrobial agent with anti-HIV activity, against herpes simplex viruses type and in vitro. Arch. Virol. 142, 2225-2235 (1997). 34. Baghian, A. & Kousoulas, K. G. Role of the Na+,K+ Pump in Herpes Simplex Type 1-Induced Cell Fusion: Melittin Causes Specific Reversion of Syncytial Mutants with the Syn1 Mutation to Syn+ (Wild-Type) Phenotype. Virology 196, 548-556 (1993). 35. Voss, T. G. et al. Alteration of intracellular potassium and sodium concentrations correlates with induction of cytopathic effects by human immunodeficiency virus. J. Virol. 70, 5447-5454 (1996). 36. Voss, T. G., Gatti, P. J., Fermin, C. D. & Garry, R. F. Reduction of Human Immunodeficiency Virus Production and Cytopathic Effects by Inhibitors of the Na+/K+/2Cl-Cotransporter. Virology 219, 291-294 (1996). 37. Belaid, A. et al. In vitro antiviral activity of dermaseptins against herpes simplex virus type 1. J. Med. Virol. 66, 229-234 (2002). 38. Lorin, C. et al. The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro. Virology 334, 264-275 (2005). 39. Robinson, W. E., Jr., McDougall, B., Tran, D. & Selsted, M. E. Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils. J. Leukoc. Biol. 63, 94-100 (1998). 194 40. Gordon, Y. J. et al. Human cathelicidin (LL-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity. Curr. Eye Res. 30, 385-394 (2005). 41. Steinstraesser, L. et al. Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides. Retrovirology. 2, (2005). 42. Jenssen, H. Anti herpes simplex virus activity of lactoferrin/lactoferricin -- an example of antiviral activity of antimicrobial protein/peptide. Cell. Mol. Life Sci. 62, 3002-3013 (2005). 43. VanCompernolle, S. E. et al. Antimicrobial peptides from amphibian skin potently inhibit human immunodeficiency virus infection and transfer of virus from dendritic cells to T cells. J. Virol. 79, 11598-11606 (2005). 44. Chiou, P. P., Lin, C. M., Perez, L. & Chen, T. T. Effect of cecropin B and a synthetic analogue on propagation of fish viruses in vitro. Mar. Biotechnol. (NY) 4, 294-302 (2002). 45. biol Matanic, V. C. & Castilla, V. Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus. Int. J Antimicrob. Agents 23, 382-389 (2004). 46. Lee, D. G. et al. Structure-antiviral activity relationships of cecropin A-magainin hybrid peptide and its analogues. J. Pept. Sci. 10, 298-303 (2004). 47. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389395 (2002). 48. Hancock, R. E. Peptide antibiotics. Lancet 349, 418-422 (1997). 49. Hancock, R. E. W. & Scott, M. G. The role of antimicrobial peptides in animal defenses. P. Natl. Acad. Sci. U. S. A. 97, 8856-8861 (2000). 50. Oren, Z. & Shai, Y. Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers 47, 451-463 (1998). 51. Andreu, D. & Rivas, L. Animal antimicrobial peptides: An overview. Biopolymers 47, 415-433 (1998). 52. Hancock, R. E. & Chapple, D. S. Peptide antibiotics. Antimicrob. Agents Chemother. 43, 1317-1323 (1999). 53. Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by [alpha]-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1462, 55-70 (1999). 195 54. Dathe, M. et al. Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry 35, 12612-12622 (1996). 55. Dathe, M. et al. General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides. Biochim. Biophys. Acta 1558, 171-186 (2002). 56. Wieprecht, T. et al. Modulation of membrane activity of amphipathic, antibacterial peptides by slight modifications of the hydrophobic moment. FEBS Lett. 417, 135-140 (1997). 57. Blondelle, S. E., Lohner, K. & Aguilar, M. I. Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity. Biochim. Biophys. Acta 1462, 89-108 (1999). 58. Chen, H. et al. Recent advances in the research and development of human defensins. Peptides In Press, Corrected Proof, (2006). 59. Ganz, T. Defensins: Antimicrobial peptides of innate immunity. Nat. Rev. Immunol. 3, 710-720 (2003). 60. Lehrer, R. I. Primate defensins. Nat. Rev. Microbiol. 2, 727-738 (2004). 61. Tang, Y. Q. et al. A Cyclic Antimicrobial Peptide Produced in Primate Leukocytes by the Ligation of Two Truncated -Defensins. Science 286, 498-502 (1999). 62. Ohta, M. et al. Mechanisms of antibacterial action of tachyplesins and polyphemusins, a group of antimicrobial peptides isolated from horseshoe crab hemocytes. Antimicrob. Agents Chemother. 36, 1460-1465 (1992). 63. Michaut, L. et al. Determination of the disulfide array of the first inducible antifungal peptide from insects: drosomycin from Drosophila melanogaster. FEBS Lett. 395, 6-10 (1996). 64. Wu, M. & Hancock, R. E. W. Interaction of the Cyclic Antimicrobial Cationic Peptide Bactenecin with the Outer and Cytoplasmic Membrane. J. Biol. Chem. 274, 29-35 (1999). 65. Oppenheim, F. G. et al. Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J. Biol. Chem. 263, 7472-7477 (1988). 66. Lawyer, C. et al. Antimicrobial activity of a 13 amino acid tryptophan-rich peptide derived from a putative porcine precursor protein of a novel family of antibacterial peptides. FEBS Lett. 390, 95-98 (1996). 196 67. Selsted, M. E. et al. Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J. Biol. Chem. 267, 4292-4295 (1992). 68. Yau, W. M., Wimley, W. C., Gawrisch, K. & White, S. H. The Preference of Tryptophan for Membrane Interfaces. Biochemistry 37, 14713-14718 (1998). 69. Persson, S., Killian, J. A. & Lindblom, G. Molecular Ordering of Interfacially Localized Tryptophan Analogs in Ester- and Ether-Lipid Bilayers Studied by 2HNMR. Biophys. J. 75, 1365-1371 (1998). 70. Halevy, R., Rozek, A., Kolusheva, S., Hancock, R. E. W. & Jelinek, R. Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay. Peptides 24, 1753-1761 (2003). 71. Schibli, D. J., Hwang, P. M. & Vogel, H. J. Structure of the Antimicrobial Peptide Tritrpticin Bound to Micelles: A Distinct Membrane-Bound Peptide Fold. Biochemistry 38, 16749-16755 (1999). 72. Rozek, A., Friedrich, C. L. & Hancock, R. E. W. Structure of the Bovine Antimicrobial Peptide Indolicidin Bound to Dodecylphosphocholine and Sodium Dodecyl Sulfate Micelles. Biochemistry 39, 15765-15774 (2000). 73. Sitaram, N., Subbalakshmi, C. & Nagaraj, R. Indolicidin, a 13-residue basic antimicrobial peptide rich in tryptophan and proline, interacts with Ca2+calmodulin. Biochem. Biophys. Res. Co. 309, 879-884 (2003). 74. McManus, A. M., Otvos, L., Hoffmann, R. & Craik, D. J. Conformational Studies by NMR of the Antimicrobial Peptide, Drosocin, and Its Non-Glycosylated Derivative: Effects of Glycosylation on Solution Conformation. Biochemistry 38, 705-714 (1999). 75. Orivel, J. et al. Ponericins, New Antibacterial and Insecticidal Peptides from the Venom of the Ant Pachycondyla goeldii. J. Biol. Chem. 276, 17823-17829 (2001). 76. Fink, J., Merrifield, R. B., Boman, A. & Boman, H. G. The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity. J. Biol. Chem. 264, 6260-6267 (1989). 77. Thennarasu, S. et al. Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A. Chem. Phys. Lipids 137, 38-51 (2005). 78. Dathe, M. & Wieprecht, T. Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim. Biophys. Acta 1462, 71-87 (1999). 79. Tossi, A., Scocchi, M., Skerlavaj, B. & Gennaro, R. Identification and characterization of a primary antibacterial domain in CAP18, a lipopolysaccharide binding protein from rabbit leukocytes. FEBS Lett. 339, 108-112 (1994). 197 80. Matsuzaki, K. et al. Modulation of Magainin 2-Lipid Bilayer Interactions by Peptide Charge. Biochemistry 36, 2104-2111 (1997). 81. Blondelle, S. E. & Houghten, R. A. Design of model amphipathic peptides having potent antimicrobial activities. Biochemistry 31, 12688-12694 (1992). 82. Kondejewski, L. H. et al. Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity. J. Biol. Chem. 274, 13181-13192 (1999). 83. Park, Y. & Hahm, K. S. Antimicrobial peptides (AMPs): peptide structure and mode of action. J. Biochem. Mol. Biol. 38, 507-516 (2005). 84. Shin, S. Y., Lee, M. K., Kim, K. L. & Hahm, K. S. Structure-antitumor and hemolytic activity relationships of synthetic peptides derived from cecropin Amagainin and cecropin A-melittin hybrid peptides. J. Pept. Res. 50, 279-285 (1997). 85. Dathe, M. et al. Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides. FEBS Lett. 403, 208-212 (1997). 86. Eisenberg, D., Schwarz, E., Komaromy, M. & Wall, R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179, 125-142 (1984). 87. Pathak, N. et al. Comparison of the effects of hydrophobicity, amphiphilicity, and alpha-helicity on the activities of antimicrobial peptides. Proteins 22, 182-186 (1995). 88. Blondelle, S. E. & Houghten, R. A. Design of model amphipathic peptides having potent antimicrobial activities. Biochemistry 31, 12688-12694 (1992). 89. Wieprecht, T. et al. Peptide hydrophobicity controls the activity and selectivity of magainin amide in interaction with membranes. Biochemistry 36, 6124-6132 (1997). 90. Blondelle, S. E. & Houghten, R. A. Probing the relationships between the structure and hemolytic activity of melittin with a complete set of leucine substitution analogs. Peptide Res. 4, 12-18. 91. Blondelle, S. E. & Houghten, R. A. Hemolytic and antimicrobial activities of the twenty-four individual omission analogues of melittin. Biochemistry 30, 46714678 (1991). 92. Chen, H. C., Brown, J. H., Morell, J. L. & Huang, C. M. Synthetic magainin analogues with improved antimicrobial activity. FEBS Lett. 236, 462-466 (1988). 198 93. Ohsaki, Y., Gazdar, A. F., Chen, H. C. & Johnson, B. E. Antitumor activity of magainin analogues against human lung cancer cell lines. Cancer Res. 52, 35343538 (1992). 94. Dathe, M. et al. Peptide Helicity and Membrane Surface Charge Modulate the Balance of Electrostatic and Hydrophobic Interactions with Lipid Bilayers and Biological Membranes. Biochemistry 35, 12612-12622 (1996). 95. Hoek, K. S., Milne, J. M., Grieve, P. A., Dionysius, D. A. & Smith, R. Antibacterial activity in bovine lactoferrin-derived peptides. Antimicrob. Agents Chemother. 41, 54-59 (1997). 96. Andersson, M., Holmgren, A. & Spyrou, G. NK-lysin, a Disulfide-containing Effector Peptide of T-lymphocytes, Is Reduced and Inactivated by Human Thioredoxin Reductase. J. Biol. Chem. 271, 10116-10120 (1996). 97. FUJII, G., Selsted, M. E. & Eisenberg, D. Defensins promote fusion and lysis of negatively charged membranes. Protein Sci. 2, 1301-1312 (1993). 98. Wimley, W. C., Selsted, M. E. & White, S. H. Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores. Protein Sci. 3, 1362-1373 (1994). 99. Matsuzaki, K. et al. Role of disulfide linkages in tachyplesin-lipid interactions. Biochemistry 32, 11704-11710 (1993). 100. Matsuzaki, K. et al. Membrane permeabilization mechanisms of a cyclic antimicrobial peptide, tachyplesin I, and its linear analog. Biochemistry 36, 97999806 (1997). 101. Li, P., Wohland, T., Ho, B. & Ding, J. L. Perturbation of Lipopolysaccharide (LPS) Micelles by Sushi (S3) Antimicrobial Peptide: The Importance of an Intermolecular Ddisulfide Bond in S3 Dimer for Bingding, Disruption, and Neutralization of LPS. J. Biol. Chem. 279, 50150-50156 (2004). 102. Wade, D. et al. All-D Amino Acid-Containing Channel-Forming Antibiotic Peptides. P. Natl. Acad. Sci. U. S. A. 87, 4761-4765 (1990). 103. Bruce alberts Molecular biology of the cell. New York, Garland Science, c2002, (2002). 104. Utsugi, T., Schroit, A. J., Connor, J., Bucana, C. D. & Fidler, I. J. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res. 51, 30623066 (1991). 105. Raetz, C. R. H. & Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635-700 (2002). 199 106. Rosenfeld, Y., Papo, N. & Shai, Y. Endotoxin (LPS) neutralization by innate immunity host-defense peptides: Peptides' properties and plausible modes of action. J. Biol. Chem. M504327200 (2005). 107. Tan, N. S. et al. Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides. FASEB J. 14, 1801-1813 (2000). 108. Tan, N. S., Ho, B. & Ding, J. L. High-affinity LPS binding domain(s) in recombinant factor C of a horseshoe crab neutralizes LPS-induced lethality. FASEB J. 14, 859-870 (2000). 109. Aurell, C. A. & Wistrom, A. O. Critical Aggregation Concentrations of GramNegative Bacterial Lipopolysaccharides (LPS). Biochem. Biophys. Res. Co. 253, 119-123 (1998). 110. Frecer, V., Ho, B. & Ding, J. L. Interpretation of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action. Eur. J. Biochem. 267, 837-852 (2000). 111. Erridge, C., nett-Guerrero, E. & Poxton, I. R. Structure and function of lipopolysaccharides. Microbes Infect. 4, 837-851 (2002). 112. Nikaido, H. & Vaara, M. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49, 1-32 (1985). 113. Santos, N. C., Silva, A. C., Castanho, M. A. R. B., Martins-Silva, J. & Saldanha, C. Evaluation of lipopolysaccharide aggregation by light scattering spectroscopy. Chembiochem 4, 96-100 (2003). 114. Falla, T. J., Karunaratne, D. N. & Hancock, R. E. W. Mode of Action of the Antimicrobial Peptide Indolicidin. J. Biol. Chem. 271, 19298-19303 (1996). 115. Piers, K. L., Brown, M. H. & Hancock, R. E. Improvement of outer membranepermeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification. Antimicrob. Agents Chemother. 38, 2311-2316 (1994). 116. Hancock, R. E. Host defence (cationic) peptides: what is their future clinical potential? Drugs 57, 469-473 (1999). 117. Matsuzaki, K., Harada, M., Funakoshi, S., Fujii, N. & Miyajima, K. Physicochemical determinants for the interactions of magainins and with acidic lipid bilayers. Biochim. Biophys. Acta 1063, 162-170 (1991). 118. Matsuzaki, K., Sugishita, K., Fujii, N. & Miyajima, K. Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. Biochemistry 34, 3423-3429 (1995). 200 119. Matsuzaki, K. Magainins as paradigm for the mode of action of pore forming polypeptides. Biochim. Biophys. Acta 1376, 391-400 (1998). 120. Egashira, M. et al. Cholesterol Modulates Interaction between an Amphipathic Class A Peptide, Ac-18A-NH2, and Phosphatidylcholine Bilayers. Biochemistry 41, 4165-4172 (2002). 121. Prenner, E. J., Lewis, R. N., Jelokhani-Niaraki, M., Hodges, R. S. & McElhaney, R. N. Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes. Biochim. Biophys. Acta 1510, 83-92 (2001). 122. Scherfeld, D., Kahya, N. & Schwille, P. Lipid Dynamics and Domain Formation in Model Membranes Composed of Ternary Mixtures of Unsaturated and Saturated Phosphatidylcholines and Cholesterol. Biophys. J. 85, 3758-3768 (2003). 123. Zhao, L. & Feng, S. S. Effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within the lipid monolayer at the air-water interface. J. Colloid Interf. Sci. In Press, Corrected Proof, (2006). 124. Epand, R. M. Lipid polymorphism and protein-lipid interactions. Biochim. Biophys. Acta 1376, 353-368 (1998). 125. Huttner, W. B. & Schmidt, A. A. Membrane curvature: a case of endofeelin'. Trends Cell Biol. 12, 155-158 (2002). 126. Matsuzaki, K. et al. Relationship of Membrane Curvature to the Formation of Pores by Magainin 2. Biochemistry 37, 11856-11863 (1998). 127. Maget-Dana, R. The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes. Biochim. Biophys. Acta 1462, 109-140 (1999). 128. Ando, S., Nishikawa, H., Takiguchi, H., Lee, S. & Sugihara, G. Antimicrobial specificity and hemolytic activity of cyclized basic amphiphilic beta-structural model peptides and their interactions with phospholipid bilayers. Biochim. Biophys. Acta 1147, 42-49 (1993). 129. Ohki, S., Marcus, E., Sukumaran, D. K. & Arnold, K. Interaction of melittin with lipid membranes. Biochim. Biophys. Acta 1194, 223-232 (1994). 130. Ambroggio, E. E., Separovic, F., Bowie, J. H., Fidelio, G. D. & Bagatolli, L. A. Direct Visualization of Membrane Leakage Induced by the Antibiotic Peptides: Maculatin, Citropin, and Aurein. Biophys. J. 89, 1874-1881 (2005). 131. Salamon, Z., Macleod, H. A. & Tollin, G. Surface plasmon resonance spectroscopy as a tool for investigating the biochemical and biophysical 201 properties of membrane protein systems. I: Theoretical principles. Biochim. Biophys. Acta 1331, 117-129 (1997). 132. Cross, G. H. et al. The metrics of surface adsorbed small molecules on the Young's fringe dual-slab waveguide interferometer. J. Phys. D Appl. Phys. 74-80 (2004). 133. Lohner, K. & Prenner, E. J. Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems. Biochim. Biophys. Acta 1462, 141-156 (1999). 134. Heller, W. T. et al. Membrane Thinning Effect of the β-Sheet Antimicrobial Protegrin. Biochemistry 39, 139-145 (2000). 135. Chen, F. Y., Lee, M. T. & Huang, H. W. Evidence for Membrane Thinning Effect as the Mechanism for Peptide-Induced Pore Formation. Biophys. J. 84, 3751-3758 (2003). 136. Mecke, A., Lee, D. K., Ramamoorthy, A., Orr, B. G. & Banaszak Holl, M. M. Membrane thinning due to antimicrobial peptide binding - An atomic force microscopy study of MSI-78 in lipid bilayers. Biophys. J. (2005). 137. Huang, H. W. Molecular mechanism of antimicrobial peptides: The origin of cooperativity. Biochim. Biophys. Acta In Press, Corrected Proof, (2006). 138. Ehrenstein, G. & Lecar, H. Electrically gated ionic channels in lipid bilayers. Q. Rev. Biophys. 10, 1-34 (1977). 139. Ben-Efraim, I., Bach, D. & Shai, Y. Spectroscopic and functional characterization of the putative transmembrane segment of the minK potassium channel. Biochemistry 32, 2371-2377 (1993). 140. Sansom, M. S. Alamethicin and related peptaibols--model ion channels. Eur. Biophys. J. 22, 105-124 (1993). 141. Pouny, Y., Rapaport, D., Mor, A., Nicolas, P. & Shai, Y. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry 31, 12416-12423 (1992). 142. Matsuzaki, K., Murase, O., Fujii, N. & Miyajima, K. An Antimicrobial Peptide, Magainin 2, Induced Rapid Flip-Flop of Phospholipids Coupled with Pore Formation and Peptide Translocation. Biochemistry 35, 11361-11368 (1996). 143. Ludtke, S. J. et al. Membrane Pores Induced by Magainin. Biochemistry 35, 13723-13728 (1996). 202 144. Pouny, Y., Rapaport, D., Mor, A., Nicolas, P. & Shai, Y. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry 31, 12416-12423 (1992). 145. Gazit, E., Boman, A., Boman, H. G. & Shai, Y. Interaction of the mammalian antibacterial peptide cecropin P1 with phospholipid vesicles. Biochemistry 34, 11479-11488 (1995). 146. Oren, Z., Lerman, J. C., Gudmundsson, G. H., Agerberth, B. & Shai, Y. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem. J. 341 ( Pt 3), 501-513 (1999). 147. Shai, Y. & Oren, Z. From "carpet" mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides. Peptides 22, 1629-1641 (2001). 148. Kondejewski, L. H. et al. Modulation of structure and antibacterial and hemolytic activity by ring size in cyclic gramicidin S analogs. J. Biol. Chem. 271, 2526125268 (1996). 149. Kondejewski, L. H. et al. Dissociation of Antimicrobial and Hemolytic Activities in Cyclic Peptide Diastereomers by Systematic Alterations in Amphipathicity. J. Biol. Chem. 274, 13181-13192 (1999). 150. Abraham, T., Lewis, R. N. A. H., Hodges, R. S. & McElhaney, R. N. Isothermal Titration Calorimetry Studies of the Binding of a Rationally Designed Analogue of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes. Biochemistry 44, 2103-2112 (2005). 151. Abraham, T., Lewis, R. N. A. H., Hodges, R. S. & McElhaney, R. N. Isothermal Titration Calorimetry Studies of the Binding of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes. Biochemistry 44, 11279-11285 (2005). 152. Oh, D. et al. NMR structural characterization of cecropin A(1-8) - magainin 2(112) and cecropin A(1-8) - melittin(1-12) hybrid peptides. J. Pept. Res. 53, 578589 (1999). 153. Merrifield, R. B. et al. Retro and Retroenantio Analogs of Cecropin-Melittin Hybrids. P. Natl. Acad. Sci. U. S. A. 92, 3449-3453 (1995). 154. Boman, H. G., Wade, D., Boman, I. A., Wahlin, B. & Merrifield, R. B. Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids. FEBS Lett. 259, 103-106 (1989). 155. Keun Kim, H. et al. Antibacterial activities of peptides designed as hybrids of antimicrobial peptides. Biotechnol. Lett. 24, 347-353 (2002). 203 156. Park, Y., Lee, D. G. & Hahm, K. S. HP(2-9)-magainin 2(1-12), a synthetic hybrid peptide, exerts its antifungal effect on Candida albicans by damaging the plasma membrane. J. Pept. Sci. 10, 204-209 (2004). 157. Oh, D. et al. Role of the Hinge Region and the Tryptophan Residue in the Synthetic Antimicrobial Peptides, Cecropin A(1-8)-Magainin 2(1-12) and Its Analogues, on Their Antibiotic Activities and Structures. Biochemistry 39, 1185511864 (2000). 158. Shin, S. Y. et al. Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells. Biochim. Biophys. Acta 1463, 209-218 (2000). 159. Cornut, I., Buttner, K., Dasseux, J. L. & Dufourcq, J. The amphipathic [alpha]helix concept : Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin. FEBS Lett. 349, 2933 (1994). 160. Javadpour, M. M. et al. De Novo Antimicrobial Peptides with Low Mammalian Cell Toxicity. J. Med. Chem. 39, 3107-3113 (1996). 161. Frecer, V., Ho, B. & Ding, J. L. De Novo Design of Potent Antimicrobial Peptides. Antimicrob. Agents Chemother. 48, 3349-3357 (2004). 162. Ge, Y. et al. In vitro antibacterial properties of pexiganan, an analog of magainin. Antimicrob. Agents Chemother. 43, 782-788 (1999). 163. Lamb, H. M. & Wiseman, L. R. Pexiganan acetate. Drugs 56, 1047-1052 (1998). 164. Sader, H. S., Fedler, K. A., Rennie, R. P., Stevens, S. & Jones, R. N. Omiganan pentahydrochloride (MBI 226), a topical 12-amino-acid cationic peptide: spectrum of antimicrobial activity and measurements of bactericidal activity. Antimicrob. Agents Chemother. 48, 3112-3118 (2004). 165. Tan, H. H. Topical antibacterial treatments for acne vulgaris : comparative review and guide to selection. Am. J. Clin. Dermatol. 5, 79-84 (2004). 166. Kavanagh, K. & Dowd, S. Histatins: antimicrobial peptides with therapeutic potential. J. Pharm. Pharmacol. 56, 285-289 (2004). 167. Chalekson, C. P., Neumeister, M. W. & Jaynes, J. Treatment of infected wounds with the antimicrobial peptide D2A21. J. Trauma. 54, 770-774 (2003). 168. Breukink, E. & de Kruijff, B. The lantibiotic nisin, a special case or not? Biochim. Biophys. Acta 1462, 223-234 (1999). 169. Moore, A. The big and small of drug discovery. Biotech versus pharma: advantages and drawbacks in drug development. EMBO Rep. 4, 114-117 (2003). 204 170. Trotti, A. et al. A multinational, randomized phase III trial of iseganan HCl oral solution for reducing the severity of oral mucositis in patients receiving radiotherapy for head-and-neck malignancy. Int. J. Radiat. Oncol. Biol. Phys. 58, 674-681 (2004). 171. Donnelly, J. P., Bellm, L. A., Epstein, J. B., Sonis, S. T. & Symonds, R. P. Antimicrobial therapy to prevent or treat oral mucositis. Lancet Infect. Dis. 3, 405-412 (2003). 172. Lewis, R. N. et al. Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes. Biochemistry 38, 15193-15203 (1999). 173. Tomasinsig, L. et al. Mechanistic and Functional Studies of the Interaction of a Proline-rich Antimicrobial Peptide with Mammalian Cells. J. Biol. Chem. 281, 383-391 (2006). 174. Papo, N. & Shai, Y. Exploring Peptide Membrane Interaction Using Surface Plasmon Resonance: Differentiation between Pore Formation versus Membrane Disruption by Lytic Peptides. Biochemistry 42, 458-466 (2003). 175. Noinville, S., Bruston, F., El Amri, C., Baron, D. & Nicolas, P. Conformation, Orientation, and Adsorption Kinetics of Dermaseptin B2 onto Synthetic Supports at Aqueous/Solid Interface. Biophys. J. 85, 1196-1206 (2003). 176. Mozsolits, H., Wirth, H. J., Werkmeister, J. & Aguilar, M. I. Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance. Biochim. Biophys. Acta 1512, 64-76 (2001). 177. Marion, D., Zasloff, M. & Bax, A. A two-dimensional NMR study of the antimicrobial peptide magainin 2. FEBS Lett. 227, 21-26 (1988). 178. Porcelli, F., Buck-Koehntop, B. A., Thennarasu, S., Ramamoorthy, A. & Veglia, G. Structures of the Dimeric and Monomeric Variants of Magainin Antimicrobial Peptides (MSI-78 and MSI-594) in Micelles and Bilayers, Determined by NMR Spectroscopy. Biochemistry 45, 5793-5799 (2006). 179. Dave, P. C., Billington, E., Pan, Y. L. & Straus, S. K. Interaction of Alamethicin with Ether-Linked Phospholipid Bilayers: Oriented Circular Dichroism, 31P Solid-State NMR, and Differential Scanning Calorimetry Studies. Biophys. J. 89, 2434-2442 (2005). 180. Magde, D., Elson, E. & Webb, W. W. Thermodynamic Fluctuations in a Reacting System; Measurement by Fluorescence Correlation Spectroscopy. Phys. Rev. Lett. 29, 705 (1972). 205 181. Widengren, J. & Rigler, R. Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces. Cell. Mol. Biol. 44, 857-879 (1998). 182. Ehrenberg, M. & Rigler, R. Rotational brownian motion and fluorescence intensify fluctuations. Chem. Phys. 4, 390-401 (1974). 183. Krichevsky, O. & Bonnet, G. Fluorescence correlation spectroscopy: the technique and its applications. Rep. Prog. Phys. 65, 251-297 (2002). 184. Magde, D., Elson, E. L. & Webb, W. W. Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13, 29-61 (1974). 185. Koppel, D. E. Statistical accuracy in fluorescence correlation spectroscopy. Phys. Rev. A 10, 1938 (1974). 186. Thompson, N. L. Topics in Fluorescence Spectroscopy. Plenum Press, New York, (1991). 187. Kim, S. A. & Schwille, P. Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience. Curr. Opin. Neurobiol. 13, 583-590 (2003). 188. Qian, H. & Elson, E. Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy. Appl. Opt. 30, 1185-1195 (1991). 189. Rigler, R., Mets, U., Widengren, J. & Kask, P. Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion. Eur. Biophys. J. 22, 169-175 (1993). 190. Wohland, T., Rigler, R. & Vogel, H. The standard deviation in fluorescence correlation spectroscopy. Biophys. J. 2987-2999 (2001). 191. Elson, E. L. & Magde, D. Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13, 1-27 (1974). 192. Kask, P., Günther, R. & Axhausen, P. Statistical accuracy in fluorescence fluctuation experiments. Eur. Biophys. J. 25, 163-169 (1997). 193. Wohland, T., Rigler, R. & Vogel, H. The Standard Deviation in Fluorescence Correlation Spectroscopy. Biophys. J. 80, 2987-2999 (2001). 194. Aragon, S. R. & Pecora, R. Fluorescence correlation spectroscopy as a probe of molecular dynamics. J. Chem. Phys. 64, 1791-1803 (1976). 195. Rigler, R., Mets, Widengren, J. & Kask, P. Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Eur. Biophys. J. 22, 169-175 (1993). 206 196. Widengren, J., Rigler, R. & Mets, l. Triplet-state monitoring by fluorescence correlation spectroscopy. J. Fluoresc. 4, 255-258 (1994). 197. Meseth, U., Wohland, T., Rigler, R. & Vogel, H. Resolution of Fluorescence Correlation Measurements. Biophys. J. 76, 1619-1631 (1999). 198. Feng, S. Interpretation of Mechanochemical Properties of Lipid Bilayer Vesicles from the Equation of State or Pressure-Area Measurement of the Monolayer at the Air-Water or Oil-Water Interface. Langmuir 15, 998-1010 (1999). 199. Clausell, A., Busquets, M. A., Pujol, M., Alsina, A. & Cajal, Y. Polymyxin Blipid interactions in Langmuir-Blodgett monolayers of Escherichia coli lipids: a thermodynamic and atomic force microscopy study. Biopolymers 75, 480-490 (2004). 200. Blume, A. A comparative study of the phase transitions of phospholipid bilayers and monolayers. Biochim. Biophys. Acta 557, 32-44 (1979). 201. Marsh, D. Lateral pressure in membranes. Biochim. Biophys. Acta 1286, 183-223 (1996). 202. Adamson, A. W. Physical Chemistry of Surfaces. Wiley and Sons, New York, (1976). 203. Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces. Wiley and Sons, New York, (1966). 204. Breslow, R. & Guo, T. Surface Tension Measurements Show That Chaotropic Salting-in Denaturants are Not Just Water-Structure Breakers. P. Natl. Acad. Sci. U. S. A. 87, 167-169 (1990). 205. Rauer, B., Neumann, E., Widengren, J. & Rigler, R. Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin [alpha]bungarotoxin with Torpedo californica acetylcholine receptor. Biophys. Chem. 58, 3-12 (1996). 206. Hink, M. A., van Hoek, A. & Visser, A. J. W. G. Dynamics of Phospholipid Molecules in Micelles: Characterization with Fluorescence Correlation Spectroscopy and Time-Resolved Fluorescence Anisotropy. Langmuir 15, 992997 (1999). 207. Krouglova, T., Vercammen, J. & Engelborghs, Y. Correct Diffusion Coefficients of Proteins in Fluorescence Correlation Spectroscopy. Application to Tubulin Oligomers Induced by Mg2+ and Paclitaxel. Biophys. J. 87, 2635-2646 (2004). 208. Wohland, T., Friedrich, K., Hovius, R. & Vogel, H. Study of Ligand-Receptor Interactions by Fluorescence Correlation Spectroscopy with Different 207 Fluorophores: Evidence That the Homopentameric 5-Hydroxytryptamine Type As Receptor Binds Only One Ligand. Biochemistry 38, 8671-8681 (1999). 209. Nakashima K & Fujimoto Y. Photoluminescent Properties of Octadecylrhodamine B in Water, in Alcohols and in Mixed Water-Alcohol Solutions. Photochem. Photobiol. 60, 563-567. 1994. 210. Hess, S. T. & Webb, W. W. Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy. Biophys. J. 83, 2300-2317 (2002). 211. Gidalevitz, D. et al. Interaction of antimicrobial peptide protegrin with biomembranes. P. Natl. Acad. Sci. U. S. A. 100, 6302-6307 (2003). 212. Pramanik, A., Thyberg, P. & Rigler, R. Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy. Chem. Phys. Lipids 104, 35-47 (2000). 213. Matsuzaki, K., Yoneyama, S. & Miyajima, K. Pore formation and translocation of melittin. Biophys. J. 73, 831-838 (1997). 214. Yang, L., Harroun, T. A., Weiss, T. M., Ding, L. & Huang, H. W. Barrel-Stave Model or Toroidal Model? A Case Study on Melittin Pores. Biophys. J. 81, 14751485 (2001). 215. Morrison, D. C. & Jacobs, D. M. Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry 13, 813-818 (1976). 216. Thomas, C. J., Surolia, N. & Surolia, A. Surface Plasmon Resonance Studies Resolve the Enigmatic Endotoxin Neutralizing Activity of Polymyxin B. J. Biol. Chem. 274, 29624-29627 (1999). 217. Roes, S., Seydel, U. & Gutsmann, T. Probing the Properties of Lipopolysaccharide Monolayers and Their Interaction with the Antimicrobial Peptide Polymyxin B by Atomic Force Microscopy. Langmuir 21, 6970-6978 (2005). 218. Tsubery, H., Ofek, I., Cohen, S., Eisenstein, M. & Fridkin, M. Modulation of the Hydrophobic Domain of Polymyxin B Nonapeptide: Effect on Outer-Membrane Permeabilization and Lipopolysaccharide Neutralization. Mol. Pharmacol. 62, 1036-1042 (2002). 219. Yu, L., Ding, J. L., Ho, B. & Wohland, T. Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: An amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity. Biochim. Biophys. Acta 1716, 29-39 (2005). 208 220. Zhao, L. Y. & Feng, S. S. Effects of lipid chain length on molecular interactions between paclitaxel and phospholipid within model biomembranes. J. Colloid Interf. Sci. 274, 55-68 (2004). 221. Crisp D.J. Surface Phenomena in Chemistry and Biology. Pergamon, London, (1958). 222. Zhao, L. Y., Feng, S. S. & Go, M. L. Investigation of molecular interactions between paclitaxel and DPPC by Langmuir film balance and differential scanning calorimetry. J. Pharm. Sci. 93, 86-98 (2004). 223. Zhao, L. Y. & Feng, S. S. Eftects of lipid chain unsaturation and headgroup type on molecular interactions between paclitaxel and phospholipid within model biomembrane. J. Colloid Interf. Sci. 285, 326-335 (2005). 224. Plenat, T. et al. Interaction of Primary Amphipathic Cell-Penetrating Peptides with Phospholipid-Supported Monolayers. Langmuir 20, 9255-9261 (2004). 225. Frecer. V. QSAR analysis of antimicrobial and haemolytic effects of cyclic cationic antimicrobial peptides derived from protegrin-1. Bioorg. Med. 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