- Email: [email protected]
The bending rigidity of phospholipid monolayers in presence of an antimicrobial frog peptide studied by X-ray grazing incidence diffraction
ARTICLE IN PRESS
Physica B 357 (2005) 185–189 www.elsevier.com/locate/physb
The bending rigidity of phospholipid monolayers in presence of an antimicrobial frog peptide studied by X-ray grazing incidence diffraction O. Konovalova,, S.M. O’Flahertya, E. Saint-Martina, G. Deutschb, E. Sevcsikb, K. Lohnerb b
a European Synchrotron Radiation Facility, 6, rue Jules Horowitz-B.P.220, 38043, Grenoble, Cedex 9, France Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Schmiedlstrasse 6, A-8042 Graz, Austria
Abstract Peptide secretion by living organisms constitutes an integral response process exploited by natural immune systems. In this work we present a model study and insight into this process reporting the thermodynamic and structural effects induced in phospholipid monolayers due to peptide insertion into the layer. Synchrotron X-ray radiation is combined with the Langmuir technique and exploited to form ‘lipid–peptide’ monolayers and probe the physical characteristics of the fundamental biological process of ‘peptide secretion’. Our experiments show that the insertion of peptides in the phospholipid layer has adverse effects on the elastic properties of the layer manifested through the bending rigidity. r 2004 Elsevier B.V. All rights reserved. PACS: 68.03.Cd; 68.18.g; 61.10.Kw Keywords: Langmuir layer; Lipid-peptide interaction; Grazing-incidence diffuse X-ray scattering; Bending rigidity
1. Introduction Antimicrobial peptide secretion is a part of the natural immune response of many living organisms [1]. These peptides allow a rapid response to infection by diverse bacterial species. For many antimicrobial peptides, it appears that their main Corresponding author.
E-mail address: [email protected] (O. Konovalov).
target is the lipid bilayer itself, rather than specific protein receptor(s) within the cell membrane [2]. An understanding of how the peptides distinguish between bacterial and mammalian cytoplasmic membranes would allow the design of novel peptide antibiotics, which could selectively kill bacteria. Despite the growing interest in these peptides, the molecular mechanism involved in antimicrobial peptide-mediated rupturing events of bacterial cell membranes still remains unclear.
0921-4526/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2004.11.053
ARTICLE IN PRESS 186
O. Konovalov et al. / Physica B 357 (2005) 185–189
Although a large number of biophysical techniques are used to study the lipid–peptide interaction, studies of the elastic properties of such systems, for example the discussion of DMPC/ magainin-2 oriented lipid layers, are rare [3]. These multilayers are comparable to lipid monolayers at the air–water interface, but have an advantage owing to the multilamellar state leading to a strong amplification of the scattering signal. However, working at full lipid hydration can present a problem. Therefore, in the present work we have investigated the effect of the antibacterial frog skin peptide PGLa on the elastic properties (bending rigidity) of phosphatidylglycerol (PG) and phosphatidylcholine (PC) monolayers. The experiments were performed with PG and PC, because PGLa was shown to discriminate between these two lipids that mimic bacterial and mammalian cytoplasmic membranes, respectively [4].
2. Experimental section 2.1. Materials Dipalmitoyl- and distearoyl-phosphatidylglycerol (DPPG and DSPG) as well as dipalmitoyl- and distearoyl-phosphatidylcholine (DPPC and DSPC) were purchased from Sigma (France) (purity499%) and used without further purification. Peptidyl–glycyl–leucine–carboxyamide (PGLa), consisting of 21 amino acids (GMASKAGAIAGKIAKVALKAL-carboxyamide [5], was purchased from Multiple Peptide Systems (San Diego, CA). The organic solvents (chloroform, methanol) used were of HPLC grade purchased from Sigma (France). 2.2. Monolayer experiments Surface pressure–area (p–A) isotherms were recorded using a commercially available Langmuir trough (Model 611A, Nima Technology Ltd.), thermostated at 20 1C. The monolayer was compressed by two moveable Teflon barriers with a velocity of 20 cm2/min. A Wilhelmy balance with 10 mm wide filter paper was used to measure the surface pressure (p) with an accuracy 70.1 mN/m.
The monolayers were prepared by spreading a defined volume of the respective pure component or mixtures of peptides with lipids onto the cleaned surface of the aqueous subphase (pure water or 10 mM Na-phosphate, pH 7.4) using a Hamilton syringe. Stock solutions of lipids and peptides were prepared in chloroform at a concentration of 0.2 mg/ml. After deposition of the samples the film was left to stand for at least 1 min before compression in order to ensure complete evaporation of the organic solvent. All isotherms were verified through repeated experimentation with fresh samples. 2.3. X-ray techniques Grazing incidence diffuse (off-specular plane) X-ray scattering (GIDXRS) experiments were performed to characterize bending rigidity of monolayers at gas/liquid interface. The experimental setup has been previously discussed [6]. The GIDXRS measurements were carried out at the Troika II (ID10B) beamline of the European Synchrotron Radiation Facility (ESRF). The monochromatic incident 8.075 keV X-ray beam (wavelength l ¼ 0.1536 nm) was first extracted from the polychromatic beam of the undulator source using a double-crystal diamond (1 1 1) monochromator. Higher harmonics were removed using two palladium-coated glass mirrors, also used to set the grazing incident angle yin to the liquid surface. yin was set to 2.025 mrad which is 76% of the critical angle for total external reflection on the vacuum/water interface. The incident beam size was 300 mm 100 mm (H V), defined using conventional slits. The resolution in horizontal plane Dq|| was defined by a vertical 300 mm wide slit 235 mm from the sample and a second vertical 500 mm slit 845 mm from the sample, directly before a vertically mounted gas filled linear position-sensitive detector (PSD). Thus, experiments essentially comprised of scanning the PSD about a vertical axis whilst simultaneously recording the scattered intensity. The vertical dependence of X-ray scattering was recorded using the PSD for qz values ranging from 0 to 5 109 m1 at different PSD position yielding q|| values ranging from 5 107 to 2 109 m1.
ARTICLE IN PRESS O. Konovalov et al. / Physica B 357 (2005) 185–189
1 kB T ; A Drg þ gq2jj þ kq4jj
3. Results and discussion The phospholipids studied in this work differ in their headgroup structure. The negatively charged phosphatidylglycerol (PG) lipids are used to mimic a bacterial cell membrane, while the zwitterionic phosphatidylcholine (PC) lipids mimic a mammalian cytoplasmic cell membrane. Phospholipids
60 50 40
10
(1)
where A is the area, g is the surface tension, k is the bending rigidity, and Dr the density difference between liquid and vapor. It should be noticed that in general g and k are dependent on q||. Expression (1) can be Fourier-transformed and consequently the height–height correlation function /z(0)z(r||)S can be extracted. Generally, it is this function that is used to calculate the differential scattering cross-section for a homogeneously fluctuating Langmuir film [11]. ds r 2 2 ~ z Þ þ sub eqz hz i / jtsc j2 rðq dO iqz Z 2 drjj ðeqz hzð0Þzðrjj Þi 1Þeiqjj rjj ; ð2Þ where tsc are the Fresnel transmission coefficients of the uncovered water surface for the scattering ~ z Þ is the Fourier angle. The form factor rðq transform of the electron density profile of the film and rsub is the electron density of the substrate (water). The X-ray measurements in the GIDXRS
30 20
0 0.4 (a)
π , mN/m
hzðqjj Þzðqjj Þi ¼
geometry are therefore sensitive to g and k through the height–height correlation function.
π , mN/m
The GIDXRS measurements were performed on monolayers prepared in an ‘‘in-house’’ custombuilt Langmuir trough especially designed for synchrotron X-ray experiments [7]. The vessel containing the trough was sealed and filled with a flow of water saturated helium to reduce water evaporation from the subphase and background due to parasitic scattering from air. All X-ray measurements were performed at 20 1C. Any rough surface produces non-zero diffuse scattering of X-rays in grazing incidence geometry [8]. The scattering intensity is defined by the height correlation spectrum of fluctuations at the surface. The liquid surface is perturbed by the thermally excited capillary waves that are determined by the surface energy associated with the deformation modes. The presence of a film of surfactants on the liquid surface causes modification of the spectrum of fluctuation due to the elastic properties of the film. Developing the free energy as a function of the mean curvature [9] the height fluctuation spectrum can be constructed and mathematically expressed as [10]
187
1.0
2 3 Area , nm2/molecule
4
60 55 50 45 40 35 30 25 20 15 10 5 0 1
(b)
0.6 0.8 Area , nm2/molecule
Fig. 1. Surface pressure–area isotherms obtained at T ¼ 20 1C on pure water: (a) DPPC (solid line) and DPPG (solid line with open circles); (b) PGLa (solid line with filled circles) and mixtures of DPPC/PGLa (solid line), DPPG/PGLa (solid line with open circles). In case of mixed samples the area is normalized to the lipid molecule.
ARTICLE IN PRESS O. Konovalov et al. / Physica B 357 (2005) 185–189
differing in their length of the aliphatic chains are used to vary the magnitude of van der Waals interaction between hydrocarbon chains. Surface pressure–area (p–A) isotherms of DPPG monolayer (Fig. 1) with PGLa at a peptide–lipid molar ratio 1:10 exhibit characteristics that differ obviously from the isotherms of the pure components. No plateau regions were observed at around 5 mN/m (coexistence range of liquid expanded and liquid condensed phase) and at 30 mN/m, where the peptide collapses. In contrast the isotherm of DPPC/PGLa mixtures can be interpreted as the sum of the isotherms of the pure components. Above the critical surface pressure ðp 30 mN=mÞ; i.e. where the peptide monolayer collapses, the DPPC/PGLa isotherm exhibits the same dependence as the isotherm of the solely DPPC monolayer. Consequently, the GIDXRS measurements were performed on monolayers both below and above this critical surface pressure. The GIDXRS measurements on monolayers of lipids only (DSPG or DSPC) exhibited a common behavior: an increase of scattering signal upon increase of surface pressure p ¼ gwaterg (decrease of surface tension) (Figs. 2 and 3). This is in agreement with the capillary model of the surface 1.8 1.6
I*Q||2, a.u.
1.4 1.2 1.0 0.8 0.6 0.4 0.2
0.3
0.4
0.5
0.6
0.7 0.8 0.9 1
Q||, nm-1 Fig. 2. Integration over qz from the GIDXRS spectra of the DSPG monolayer at p ¼ 15 mN/m (open circle) and p ¼ 30 mN/m (filed circle) compared with the spectra of the DSPG/PGLa monolayer at the same surface pressures: p ¼ 15 mN/m (open triangle) and p ¼ 30 mN/m (filled triangle). Measurements are performed at T ¼ 20 1C on a subphase with 10 mM Na-phosphate, pH 7.4.
1.8 1.6 1.4
I*Q||2, a.u.
188
1.2 1.0 0.8 0.6 0.4 0.2
0.3
0.4
0.5
0.6
0.7 0.8 0.9 1
Q||, nm-1 Fig. 3. Integration over qz from the GIDXRS spectra of the DSPC monolayer at p ¼ 15 mN/m (open circle) and p ¼ 30 mN/m (filed circle) compared with the spectra of the DSPC/PGLa monolayer at the same surface pressures: p ¼ 15 mN/m (open triangle) and p ¼ 30 mN/m (filled triangle). Measurements are performed at T ¼ 20 1C on a subphase with 10 mM Na-phosphate, pH 7.4.
perturbation summarized in formulas (1) and (2). Upon adding peptides the situation changed dramatically. The DSPG/PGLa system exhibits a decrease of scattering signal compared to the film of solely lipids at the same surface pressure (Fig. 2). This clearly indicates an increase of bending rigidity of the monolayer that reduces the height fluctuations of the surface, which is in agreement with earlier observations [3]. Thereby, it was found that the presence of the peptide destroys the integrity of the DSPG monolayer (chain order) demonstrating that the peptide penetrates into the lipid-chain region. Scattering from the DSPC/ PGla monolayer exhibits opposite characteristics (Fig. 3). Upon addition of peptides the curves shift upwards with respect to the curves measured without PGLa. This indicates a reduction of the overall bending rigidity of the monolayer. From our previous studies we know that DSPC does not interact with the peptide molecules at a molecular level and forms separated domains on the air/ water interface [7]. The PGLa molecules are surface active [7] and occupy the available surface area. Our control measurements demonstrated that the diffuse scattering signal form air/water interface increases in the presence of PGLa (Fig. 4). This can be explained considering a
ARTICLE IN PRESS O. Konovalov et al. / Physica B 357 (2005) 185–189
189
Acknowledgments
2.2 2.0
K.L. acknowledges financial support from the Commission of the European Communities, specific RTD programme ‘‘Quality of Life and Management of Living Resources’’, QLK2-CT2002-01001 ‘‘Antimicrobial endotoxin neutralizing peptides to combat infectious diseases’’.
I*Q||2, a.u.
1.8 1.6 1.4 1.2 1.0 0.8 0.6
References
0.4 0.2
0.3
0.4
0.5
0.6
0.7 0.8 0.9 1
Q||, nm-1 Fig. 4. Integration over qz from the GIDXRS spectra of the PGLa monolayer at p ¼ 15 mN/m (open circle) compared with the spectra taken from the pure subphase without a monolayer (filled circle). Measurements are performed at T ¼ 20 1C, subphase 10 mM Na-phosphate, pH 7.4.
reduction of surface tension. Therefore, owing to the phase separation of DSPC and PGLa an averaged effect is measured. Quantitative analysis of the curves is in progress and will be discussed anon. One can envisage that changes of membrane bending rigidity owing to peptide insertion into the lipid bilayer result in domain and/or defect structures. This in turn may affect the membrane integrity and lead to dysfunction or in the worst case to membrane disruption.
[1] M. Zasloff, Nature 415 (2002) 389. [2] K. Lohner, in: K. Lohner (Ed.), Development of Novel Antimicrobial Agents: Emerging Strategies, Horizon Scientific Press, Wymondham, Norfolk, UK, 2001, pp. 149–165. [3] T. Salditt, Curr. Opin. Struct. Biol. 13 (2003) 467. [4] A. Latal, G. Degovics, R.F. Epand, R.M. Epand, K. Lohner, Eur. J. Biochem. 248 (1997) 938. [5] D. Andreu, H. Aschauer, G. Kreil, R.B. Merrifield, Eur. J. Biochem. 149 (1985) 531. [6] S. Mora, J. Daillant, K. Mecke, D. Luzet, A. Braslau, M. Alba, B. Struth, Phys. Rev. Lett. 90 (2003) 216101. [7] O. Konovalov, I. Myagkov, B. Struth, K. Lohner, Eur. Biophys. J. 31 (2002) 428. [8] S.K. Sinha, E.B. Sirota, S. Garoff, H.B. Stanley, Phys. Rev. B 38 (1988) 2297. [9] W. Helfrich, Z. Naturforsch. 28c (1973) 693. [10] C. Gourier J. Daillant, A. Braslau, M. Alba, K. Quinn, D. Luzet, C. Blot, D. Chatenay, G. Gru¨bel, J.-F. Legrand, G. Vignaud, Phys. Rev. Lett. 78 (1997) 3157. [11] S. Mora, J. Daillant, D. Luzet, B. Struth, Europhys. Lett. 66 (2004) 694.
Physica B 357 (2005) 185–189 www.elsevier.com/locate/physb
The bending rigidity of phospholipid monolayers in presence of an antimicrobial frog peptide studied by X-ray grazing incidence diffraction O. Konovalova,, S.M. O’Flahertya, E. Saint-Martina, G. Deutschb, E. Sevcsikb, K. Lohnerb b
a European Synchrotron Radiation Facility, 6, rue Jules Horowitz-B.P.220, 38043, Grenoble, Cedex 9, France Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Schmiedlstrasse 6, A-8042 Graz, Austria
Abstract Peptide secretion by living organisms constitutes an integral response process exploited by natural immune systems. In this work we present a model study and insight into this process reporting the thermodynamic and structural effects induced in phospholipid monolayers due to peptide insertion into the layer. Synchrotron X-ray radiation is combined with the Langmuir technique and exploited to form ‘lipid–peptide’ monolayers and probe the physical characteristics of the fundamental biological process of ‘peptide secretion’. Our experiments show that the insertion of peptides in the phospholipid layer has adverse effects on the elastic properties of the layer manifested through the bending rigidity. r 2004 Elsevier B.V. All rights reserved. PACS: 68.03.Cd; 68.18.g; 61.10.Kw Keywords: Langmuir layer; Lipid-peptide interaction; Grazing-incidence diffuse X-ray scattering; Bending rigidity
1. Introduction Antimicrobial peptide secretion is a part of the natural immune response of many living organisms [1]. These peptides allow a rapid response to infection by diverse bacterial species. For many antimicrobial peptides, it appears that their main Corresponding author.
E-mail address: [email protected] (O. Konovalov).
target is the lipid bilayer itself, rather than specific protein receptor(s) within the cell membrane [2]. An understanding of how the peptides distinguish between bacterial and mammalian cytoplasmic membranes would allow the design of novel peptide antibiotics, which could selectively kill bacteria. Despite the growing interest in these peptides, the molecular mechanism involved in antimicrobial peptide-mediated rupturing events of bacterial cell membranes still remains unclear.
0921-4526/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2004.11.053
ARTICLE IN PRESS 186
O. Konovalov et al. / Physica B 357 (2005) 185–189
Although a large number of biophysical techniques are used to study the lipid–peptide interaction, studies of the elastic properties of such systems, for example the discussion of DMPC/ magainin-2 oriented lipid layers, are rare [3]. These multilayers are comparable to lipid monolayers at the air–water interface, but have an advantage owing to the multilamellar state leading to a strong amplification of the scattering signal. However, working at full lipid hydration can present a problem. Therefore, in the present work we have investigated the effect of the antibacterial frog skin peptide PGLa on the elastic properties (bending rigidity) of phosphatidylglycerol (PG) and phosphatidylcholine (PC) monolayers. The experiments were performed with PG and PC, because PGLa was shown to discriminate between these two lipids that mimic bacterial and mammalian cytoplasmic membranes, respectively [4].
2. Experimental section 2.1. Materials Dipalmitoyl- and distearoyl-phosphatidylglycerol (DPPG and DSPG) as well as dipalmitoyl- and distearoyl-phosphatidylcholine (DPPC and DSPC) were purchased from Sigma (France) (purity499%) and used without further purification. Peptidyl–glycyl–leucine–carboxyamide (PGLa), consisting of 21 amino acids (GMASKAGAIAGKIAKVALKAL-carboxyamide [5], was purchased from Multiple Peptide Systems (San Diego, CA). The organic solvents (chloroform, methanol) used were of HPLC grade purchased from Sigma (France). 2.2. Monolayer experiments Surface pressure–area (p–A) isotherms were recorded using a commercially available Langmuir trough (Model 611A, Nima Technology Ltd.), thermostated at 20 1C. The monolayer was compressed by two moveable Teflon barriers with a velocity of 20 cm2/min. A Wilhelmy balance with 10 mm wide filter paper was used to measure the surface pressure (p) with an accuracy 70.1 mN/m.
The monolayers were prepared by spreading a defined volume of the respective pure component or mixtures of peptides with lipids onto the cleaned surface of the aqueous subphase (pure water or 10 mM Na-phosphate, pH 7.4) using a Hamilton syringe. Stock solutions of lipids and peptides were prepared in chloroform at a concentration of 0.2 mg/ml. After deposition of the samples the film was left to stand for at least 1 min before compression in order to ensure complete evaporation of the organic solvent. All isotherms were verified through repeated experimentation with fresh samples. 2.3. X-ray techniques Grazing incidence diffuse (off-specular plane) X-ray scattering (GIDXRS) experiments were performed to characterize bending rigidity of monolayers at gas/liquid interface. The experimental setup has been previously discussed [6]. The GIDXRS measurements were carried out at the Troika II (ID10B) beamline of the European Synchrotron Radiation Facility (ESRF). The monochromatic incident 8.075 keV X-ray beam (wavelength l ¼ 0.1536 nm) was first extracted from the polychromatic beam of the undulator source using a double-crystal diamond (1 1 1) monochromator. Higher harmonics were removed using two palladium-coated glass mirrors, also used to set the grazing incident angle yin to the liquid surface. yin was set to 2.025 mrad which is 76% of the critical angle for total external reflection on the vacuum/water interface. The incident beam size was 300 mm 100 mm (H V), defined using conventional slits. The resolution in horizontal plane Dq|| was defined by a vertical 300 mm wide slit 235 mm from the sample and a second vertical 500 mm slit 845 mm from the sample, directly before a vertically mounted gas filled linear position-sensitive detector (PSD). Thus, experiments essentially comprised of scanning the PSD about a vertical axis whilst simultaneously recording the scattered intensity. The vertical dependence of X-ray scattering was recorded using the PSD for qz values ranging from 0 to 5 109 m1 at different PSD position yielding q|| values ranging from 5 107 to 2 109 m1.
ARTICLE IN PRESS O. Konovalov et al. / Physica B 357 (2005) 185–189
1 kB T ; A Drg þ gq2jj þ kq4jj
3. Results and discussion The phospholipids studied in this work differ in their headgroup structure. The negatively charged phosphatidylglycerol (PG) lipids are used to mimic a bacterial cell membrane, while the zwitterionic phosphatidylcholine (PC) lipids mimic a mammalian cytoplasmic cell membrane. Phospholipids
60 50 40
10
(1)
where A is the area, g is the surface tension, k is the bending rigidity, and Dr the density difference between liquid and vapor. It should be noticed that in general g and k are dependent on q||. Expression (1) can be Fourier-transformed and consequently the height–height correlation function /z(0)z(r||)S can be extracted. Generally, it is this function that is used to calculate the differential scattering cross-section for a homogeneously fluctuating Langmuir film [11]. ds r 2 2 ~ z Þ þ sub eqz hz i / jtsc j2 rðq dO iqz Z 2 drjj ðeqz hzð0Þzðrjj Þi 1Þeiqjj rjj ; ð2Þ where tsc are the Fresnel transmission coefficients of the uncovered water surface for the scattering ~ z Þ is the Fourier angle. The form factor rðq transform of the electron density profile of the film and rsub is the electron density of the substrate (water). The X-ray measurements in the GIDXRS
30 20
0 0.4 (a)
π , mN/m
hzðqjj Þzðqjj Þi ¼
geometry are therefore sensitive to g and k through the height–height correlation function.
π , mN/m
The GIDXRS measurements were performed on monolayers prepared in an ‘‘in-house’’ custombuilt Langmuir trough especially designed for synchrotron X-ray experiments [7]. The vessel containing the trough was sealed and filled with a flow of water saturated helium to reduce water evaporation from the subphase and background due to parasitic scattering from air. All X-ray measurements were performed at 20 1C. Any rough surface produces non-zero diffuse scattering of X-rays in grazing incidence geometry [8]. The scattering intensity is defined by the height correlation spectrum of fluctuations at the surface. The liquid surface is perturbed by the thermally excited capillary waves that are determined by the surface energy associated with the deformation modes. The presence of a film of surfactants on the liquid surface causes modification of the spectrum of fluctuation due to the elastic properties of the film. Developing the free energy as a function of the mean curvature [9] the height fluctuation spectrum can be constructed and mathematically expressed as [10]
187
1.0
2 3 Area , nm2/molecule
4
60 55 50 45 40 35 30 25 20 15 10 5 0 1
(b)
0.6 0.8 Area , nm2/molecule
Fig. 1. Surface pressure–area isotherms obtained at T ¼ 20 1C on pure water: (a) DPPC (solid line) and DPPG (solid line with open circles); (b) PGLa (solid line with filled circles) and mixtures of DPPC/PGLa (solid line), DPPG/PGLa (solid line with open circles). In case of mixed samples the area is normalized to the lipid molecule.
ARTICLE IN PRESS O. Konovalov et al. / Physica B 357 (2005) 185–189
differing in their length of the aliphatic chains are used to vary the magnitude of van der Waals interaction between hydrocarbon chains. Surface pressure–area (p–A) isotherms of DPPG monolayer (Fig. 1) with PGLa at a peptide–lipid molar ratio 1:10 exhibit characteristics that differ obviously from the isotherms of the pure components. No plateau regions were observed at around 5 mN/m (coexistence range of liquid expanded and liquid condensed phase) and at 30 mN/m, where the peptide collapses. In contrast the isotherm of DPPC/PGLa mixtures can be interpreted as the sum of the isotherms of the pure components. Above the critical surface pressure ðp 30 mN=mÞ; i.e. where the peptide monolayer collapses, the DPPC/PGLa isotherm exhibits the same dependence as the isotherm of the solely DPPC monolayer. Consequently, the GIDXRS measurements were performed on monolayers both below and above this critical surface pressure. The GIDXRS measurements on monolayers of lipids only (DSPG or DSPC) exhibited a common behavior: an increase of scattering signal upon increase of surface pressure p ¼ gwaterg (decrease of surface tension) (Figs. 2 and 3). This is in agreement with the capillary model of the surface 1.8 1.6
I*Q||2, a.u.
1.4 1.2 1.0 0.8 0.6 0.4 0.2
0.3
0.4
0.5
0.6
0.7 0.8 0.9 1
Q||, nm-1 Fig. 2. Integration over qz from the GIDXRS spectra of the DSPG monolayer at p ¼ 15 mN/m (open circle) and p ¼ 30 mN/m (filed circle) compared with the spectra of the DSPG/PGLa monolayer at the same surface pressures: p ¼ 15 mN/m (open triangle) and p ¼ 30 mN/m (filled triangle). Measurements are performed at T ¼ 20 1C on a subphase with 10 mM Na-phosphate, pH 7.4.
1.8 1.6 1.4
I*Q||2, a.u.
188
1.2 1.0 0.8 0.6 0.4 0.2
0.3
0.4
0.5
0.6
0.7 0.8 0.9 1
Q||, nm-1 Fig. 3. Integration over qz from the GIDXRS spectra of the DSPC monolayer at p ¼ 15 mN/m (open circle) and p ¼ 30 mN/m (filed circle) compared with the spectra of the DSPC/PGLa monolayer at the same surface pressures: p ¼ 15 mN/m (open triangle) and p ¼ 30 mN/m (filled triangle). Measurements are performed at T ¼ 20 1C on a subphase with 10 mM Na-phosphate, pH 7.4.
perturbation summarized in formulas (1) and (2). Upon adding peptides the situation changed dramatically. The DSPG/PGLa system exhibits a decrease of scattering signal compared to the film of solely lipids at the same surface pressure (Fig. 2). This clearly indicates an increase of bending rigidity of the monolayer that reduces the height fluctuations of the surface, which is in agreement with earlier observations [3]. Thereby, it was found that the presence of the peptide destroys the integrity of the DSPG monolayer (chain order) demonstrating that the peptide penetrates into the lipid-chain region. Scattering from the DSPC/ PGla monolayer exhibits opposite characteristics (Fig. 3). Upon addition of peptides the curves shift upwards with respect to the curves measured without PGLa. This indicates a reduction of the overall bending rigidity of the monolayer. From our previous studies we know that DSPC does not interact with the peptide molecules at a molecular level and forms separated domains on the air/ water interface [7]. The PGLa molecules are surface active [7] and occupy the available surface area. Our control measurements demonstrated that the diffuse scattering signal form air/water interface increases in the presence of PGLa (Fig. 4). This can be explained considering a
ARTICLE IN PRESS O. Konovalov et al. / Physica B 357 (2005) 185–189
189
Acknowledgments
2.2 2.0
K.L. acknowledges financial support from the Commission of the European Communities, specific RTD programme ‘‘Quality of Life and Management of Living Resources’’, QLK2-CT2002-01001 ‘‘Antimicrobial endotoxin neutralizing peptides to combat infectious diseases’’.
I*Q||2, a.u.
1.8 1.6 1.4 1.2 1.0 0.8 0.6
References
0.4 0.2
0.3
0.4
0.5
0.6
0.7 0.8 0.9 1
Q||, nm-1 Fig. 4. Integration over qz from the GIDXRS spectra of the PGLa monolayer at p ¼ 15 mN/m (open circle) compared with the spectra taken from the pure subphase without a monolayer (filled circle). Measurements are performed at T ¼ 20 1C, subphase 10 mM Na-phosphate, pH 7.4.
reduction of surface tension. Therefore, owing to the phase separation of DSPC and PGLa an averaged effect is measured. Quantitative analysis of the curves is in progress and will be discussed anon. One can envisage that changes of membrane bending rigidity owing to peptide insertion into the lipid bilayer result in domain and/or defect structures. This in turn may affect the membrane integrity and lead to dysfunction or in the worst case to membrane disruption.
[1] M. Zasloff, Nature 415 (2002) 389. [2] K. Lohner, in: K. Lohner (Ed.), Development of Novel Antimicrobial Agents: Emerging Strategies, Horizon Scientific Press, Wymondham, Norfolk, UK, 2001, pp. 149–165. [3] T. Salditt, Curr. Opin. Struct. Biol. 13 (2003) 467. [4] A. Latal, G. Degovics, R.F. Epand, R.M. Epand, K. Lohner, Eur. J. Biochem. 248 (1997) 938. [5] D. Andreu, H. Aschauer, G. Kreil, R.B. Merrifield, Eur. J. Biochem. 149 (1985) 531. [6] S. Mora, J. Daillant, K. Mecke, D. Luzet, A. Braslau, M. Alba, B. Struth, Phys. Rev. Lett. 90 (2003) 216101. [7] O. Konovalov, I. Myagkov, B. Struth, K. Lohner, Eur. Biophys. J. 31 (2002) 428. [8] S.K. Sinha, E.B. Sirota, S. Garoff, H.B. Stanley, Phys. Rev. B 38 (1988) 2297. [9] W. Helfrich, Z. Naturforsch. 28c (1973) 693. [10] C. Gourier J. Daillant, A. Braslau, M. Alba, K. Quinn, D. Luzet, C. Blot, D. Chatenay, G. Gru¨bel, J.-F. Legrand, G. Vignaud, Phys. Rev. Lett. 78 (1997) 3157. [11] S. Mora, J. Daillant, D. Luzet, B. Struth, Europhys. Lett. 66 (2004) 694.