Second Harmonic Generation to Monitor the Interactions of the Antimicrobial Mycosubtilin with Membrane-Mimicking Interfacial Monolayers
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- Nasir, M.N., Benichou, E., Guez, J.S. et al. BioNanoSci. (2012) 2: 108. doi:10.1007/s12668-012-0037-6
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Mycosubtilin is a strong antimicrobial agent belonging to the iturinic lipopeptide family which contains a single tyrosine residue. Its cell target has been shown to be the cytoplasmic membrane. This tyrosine residue has been previously shown to be essential for the biological activity of mycosubtilin. Since we have previously demonstrated that tyrosine, an aromatic amino acid, can be used as an endogenous probe for the frequency doubling process, the presence of a tyrosine residue in mycosubtilin allowed us to investigate the interactions of mycosubtilin with biomimetic lipid monolayers at the air–water interface by second harmonic generation (SHG). Mycosubtilin was added underneath dipalmitoylphosphatidylcholine or cholesterol monolayers at the air–water interface and significant increases in the surface pressure were observed in both cases. This observation demonstrates that mycosubtilin interacts with these biomimetic membranes. A light polarization resolved analysis of the SHG signals recovered for these two systems was then performed and confirmed that those interactions between the tyrosine residue in mycosubtilin and the membranes could be monitored by SHG. Furthermore, the differences exhibited by the nonlinear optical measurements for different membranes showed that these interactions depend on the nature of the biomimetic membrane present at the air–water interface.
KeywordsMycosubtilinBiomimetic membranesSecond harmonic generationIturinAntimicrobial peptide
Second harmonic generation (SHG) is the nonlinear optical process whereby two photons at a fundamental frequency are converted into one photon at the harmonic frequency. SHG has been shown to be an ideal tool to investigate molecular organization at the nanometer scale owing to its cancellation in centrosymmetric media like liquids within the electric dipole approximation. Hence, SHG has been applied in the past for the characterization of many different systems at liquid interfaces [1, 2]. Besides, recent studies aiming at extending its use to proteins and peptides in the absence of exogenous molecular probes have been recently performed. In particular, they demonstrated that aromatic amino acids like tryptophan (Trp) and tyrosine (Tyr) can be used as endogenous probes of proteins and peptides. The first hyperpolarizability of Trp and Tyr, namely their cross-section for the SHG process, and that of a short tripeptide containing two lysines and one Trp, were indeed determined by hyper Rayleigh scattering, an incoherent SHG process, in the bulk of an aqueous phase . These measurements therefore showed that it is possible to devise experiments targeting Trp and Tyr residues in short synthetic peptides using SHG at the air–water interface [4, 5]. These results confirmed the previous works reported for Trp derivative at interfaces and extended them to the case of Tyr [6, 7]. As a result, through a detailed analysis of the nonlinear optical response, in particular when selecting the polarization state of the incoming and outgoing fields, a molecular picture of the interface can be retrieved, a powerful tool to investigate the interaction of Trp- and Tyr-rich peptides with phospholipids [2, 8].
In the present work, we perform SHG experiments probing at the air–water interface the presence of a biologically active peptide containing a single Tyr residue. Indeed, mycosubtilin is constituted by the heptapeptide L-Asn→D-Tyr→D-Asn→L-Gln→L-Pro→D-Ser→L-Asn associated with a β-amino fatty acid residue . Mycosubtilin is a strong antimicrobial cyclolipopeptide produced by Bacillus subtilis strains and involved in the biological control of plant diseases [10, 11]. Its cell target has been shown to be the cytoplasmic membrane . To model the interaction between the lipopeptide and the biological membranes, its interfacial properties and its adsorption to various lipid monolayers at the air–water interface have been extensively studied and it has been shown that cholesterol is a preferential partner of mycosubtilin when it interacts with biomimetic membranes [13–15]. We therefore investigated the adsorption of mycosubtilin to interfacial monolayers constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol by simultaneously measuring the SHG signal and the surface pressure by standard tensiometry.
2 Materials and Methods
Cholesterol (Chol) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, purchased from Sigma Chemical (St. Louis, MO), were dissolved in hexane–ethanol (9/1, v/v). Mycosubtilin, prepared as described previously , was dissolved in dimethylsulfoxide.
2.2 Film Formation and Surface Pressure Measurements
Adsorption experiments were performed at 18°C using a small Teflon dish (4 cm diameter, 12 mL volume) and a pure water subphase (resistivity of 18.2 MΩ·cm). This low temperature was required for the optical experiments to prevent evaporation during the course of the measurements. The lipid monolayers were prepared by deposition of the DPPC or Chol solutions at the air–water interface, thereby setting an initial surface pressure Πi of 10 mN/m. After stabilization of the lipid monolayer, mycosubtilin was injected into the subphase at a final concentration of 1.1 μM. To avoid fluctuations in the SHG optical measurements, the subphase was stirred only during the first minutes after the lipopeptide injection.
2.3 Second Harmonic Generation Measurements
The SHG setup was based on a femtosecond Ti-sapphire oscillator laser source providing pulses with duration of about 70 fs at a repetition rate of 80 MHz (Spectra-Physics) [17, 18]. After passing through a low-pass filter to remove any unwanted harmonic light generated prior to the interface, the fundamental beam set to a wavelength of 800 nm and an averaged power of about 1 W was focused by a lens with a 10-cm focal length onto the air–water interface. The incidence angle was set at a value of 70° corresponding to an optimum incidence angle for the SHG intensity in reflection. The SH light was collected by a lens with a 10-cm focal length and separated from its fundamental counterpart by a high-pass filter. The SH light was detected with a water-cooled back-illuminated CCD camera (Andor) placed after a spectrometer (Jobin-Yvon). The fundamental input beam was linearly polarized and the input polarization angle γ was selected with a rotating half-wave plate. The angle γ = 0 corresponds to a p-polarized fundamental beam and γ = π/2 to an s-polarized fundamental beam. An analyzer, placed in front of the spectrometer, was used to separate the S- and P-polarized SH intensities. In all the kinetic experiments, we used a p-in and P-out polarization configuration.
3 Results and Discussion
3.1 Interaction of Mycosubtilin with the DPPC Monolayer
Values obtained for the χxxz/χzzz and χzxx/χzzz ratios from the adjustment to Eq. 1a
Neat air/water interfacea
Pure DPPC monolayer
Mixed MS-DPPC monolayer
Pure cholesterol monolayer
Mixed MS-cholesterol monolayer
3.2 Interaction of Mycosubtilin with Cholesterol Monolayer
To test whether these changes are the signature of the interaction of the lipopeptide with cholesterol, mycosubtilin was injected under the Chol monolayer at surface pressures of 3 and 30 mN/m. Light polarization curves of the Chol-mycosubtilin monolayers were then recorded after complete adsorption of the lipopeptide, i.e., when the surface pressure change ΔΠ was stabilized at ~25 mN/m for the surface pressure Πi of 3 mN/m or stabilized at ~10 mN/m for the surface pressure Πi of 30 mN/m. In both cases, the P-polarized curves exhibited the two weak peaks at the fundamental polarization angles 90° and 270° besides the two maxima obtained at 0° and 180° (data not shown). In both cases, the complete disappearance of the S-polarized curve was observed. Using Eq. 1a, the elements of the susceptibility tensor were determined in the case of a pure cholesterol monolayer and a mixed mycosubtilin-cholesterol monolayer, see Table 1. For the pure cholesterol monolayer, the extracted value for the χzxx/χzzz ratio is similar to that obtained in the case of a DPPC monolayer whereas a greater value is obtained for the χxxz/χzzz ratio. As for the DPPC monolayer, the presence of mycosubtilin induced a dramatic drop of the two elements χzxx and χxxz. The χxxz element vanishes as expected for a vanishing S-polarized intensity curve. Similarly, the absolute value of the ratio χzxx/χzzz also decreased as compared to the value obtained in the case of the pure cholesterol monolayer. However, this time the decrease is accompanied by a relative phase change. The latter change is associated with the response of the Tyr residue.
In conclusion, we have demonstrated that, while the SHG signals from the DPPC and Chol monolayers have not the same intensity, both give similar SHG light polarization curves. This similarity was not preserved when mycosubtilin interacted with the DPPC and the cholesterol monolayers. The mycosubtilin-induced changes in the SHG light polarization curves are however more pronounced in the case of a Chol monolayer as compared to the case of the DPPC monolayer. This might be related to the different orientation of mycosubtilin when it is inserted into DPPC or Chol monolayers.
Hence, SHG is demonstrated to be a powerful tool to investigate Tyr containing peptides, similarly to Trp containing peptides. It is also sensitive enough to monitor peptides containing a single Tyr residue. Further work will be carried on to extract the molecular parameters from these results and conclude further on these interactions.