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Membrane Orientation of MSI-78 Measured by Sum Frequency Generation Vibrational Spectroscopy Pei Yang, Ayyalusamy Ramamoorthy, and Zhan Chen* Biophysics and Department of Chemistry, 930 North University Avenue, University of Michigan, Ann Arbor, Michigan 48109, United States ABSTRACT: Antimicrobial peptides (AMPs) selectively disrupt bacterial cell membranes to kill bacteria whereas they either do not or weakly interact with mammalian cells. The orientations of AMPs in lipid bilayers mimicking bacterial and mammalian cell membranes are related to their antimicrobial activity and selectivity. To understand the role of AMPlipid interactions in the functional properties of AMPs better, we determined the membrane orientation of an AMP (MSI-78 or pexiganan) in various model membranes using sum frequency generation (SFG) vibrational spectroscopy. A solid-supported single 1,2-dipalmitoyl-an-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) bilayer or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) bilayer was used as a model bacterial cell membrane. A supported 1,2-dipalmitoyl-an-glycero-3-phosphocholine (DPPC) bilayer or a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was used as a model mammalian cell membrane. Our SFG results indicate that the helical MSI-78 molecules are associated with the bilayer surface with ∼70° deviation from the bilayer normal in the negatively charged gel-phase DPPG bilayer at 400 nM peptide concentration. However, when the concentration was increased to 600 nM, MSI-78 molecules changed their orientation to make a 25° tilt from the lipid bilayer normal whereas multiple orientations were observed for an even higher peptide concentration in agreement with toroidal-type pore formation as reported in a previous solid-state NMR study. In contrary, no interaction between MSI-78 and a zwitterionic DPPC bilayer was observed even at a much higher peptide concentration (∼12 000 nM). These results demonstrate that SFG can provide insights into the antibacterial activity and selectivity of MSI-78. Interestingly, the peptide exhibits a concentration-dependent membrane orientation in the lamellar-phase POPG bilayer and was also found to induce toroidal-type pore formation. The deduced lipid flip-flop from SFG signals observed from lipids also supports MSI-78induced toroidal-type pore formation.
1. INTRODUCTION Various peptides have been designed and tested as antimicrobial compounds to prevent bacterial drug resistance.1,2 It has been widely shown that antimicrobial peptides (AMPs) can differentiate mammalian cell membranes from bacteria cell membranes and selectively kill bacteria without harming mammalian cells.37 An ideal AMP should have high efficacy in killing bacterial cells and no hemolytic activity. An examination of the detailed structural information of AMPs associated with various cell membranes can lead to a further understanding of peptide membrane interactions, aiding in the design and development of AMPs with improved properties. Bacterial cell membranes contain ∼25% negatively charged lipids, and a mammalian cell membrane usually consists of zwitterionic lipids such as phosphatidylcholine (PC). The electrostatic attraction between negatively charged lipids (such as 1-palmitoyl-2-oleoylsn-glycero-3-[phospho-rac-(1-glycerol)] or POPG) and a positively charged AMP is believed to be responsible for the binding of AMPs to bacterial cell membranes but not the mammalian ones. To avoid the complexity of the native cell membrane, substrate-supported lipid bilayers have been commonly used as models for the cell membrane. 813 In this study, we used such model membranes to investigate AMPmembrane interactions. r 2011 American Chemical Society
Many AMPs kill bacteria by disrupting their cell membranes. Several modes of action of AMPs have been proposed: barrel stave, toroidal pore, carpetlike, and others.5,7,1417 In these different modes of action, AMPs adopt different orientations relative to the membrane surface. Therefore, important information regarding the AMP mode of action can be deduced by determining the membrane orientation of an AMP. Recently, a nonlinear optical spectroscopic technique, sum frequency generation (SFG) vibrational spectroscopy, has been successfully applied to determine the membrane orientation of peptides (including AMPs) and proteins.1826 SFG provides the vibrational spectra of surfaces and interfaces with a submonolayer surface specificity.2746 It requires only a very small amount of sample and can probe surfaces and interfaces in real time. NMR techniques have been widely used to investigate the molecular interactions between AMPs and model cell membranes, and excellent results have been obtained in such studies.4754 Unlike NMR, which uses premixed multilayer lipidpeptide mixtures, SFG can probe interactions between AMPs and a single supported lipid bilayer in situ in real time. Received: March 20, 2011 Revised: May 9, 2011 Published: May 19, 2011 7760
dx.doi.org/10.1021/la201048t | Langmuir 2011, 27, 7760–7767
Langmuir Scheme 1. (Top to bottom) Molecular Structures of 1,2Dipalmitoyl-an-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), 1,2-Dipalmitoyl-an-glycero-3-phosphocholine (DPPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1glycerol)] (POPG), and 1-Palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC)
MSI-78 (also known as pexiganan) is a designed synthetic peptide that is an analog of magainin 2 but with a much higher antimicrobial activity and selectivity.54,55 Previous research on MSI-78 reported that it adopts an R-helical structure in a membrane environment. The orientation of MSI-78 molecules in membranes studied by using various techniques including NMR54,5658 revealed that the amphipathic helical peptide is oriented on the surface of the lipid bilayer. Although these results are exciting and provide insights into the function of MSI-78, NMR studies typically require approximately milligram quantities of the peptide. However, there is significant interest in understanding the peptidemembrane interactions at a lower peptide concentration, and experimental data under such conditions are unavailable for MSI-78. For example, experimental measurements from lipid bilayer samples containing a peptide concentration at the minimum inhibitory concentration (MIC) or lower will provide physiologically relevant results. In this article, we report SFG experimental results on MSI-78 embedded in model bacterial and mammalian cell membranes. We determined the membrane orientations of MSI-78 in various lipid bilayers. Our results further reveal that MSI-78 can strongly interact with model bacterial cell membranes and induce strong disruptions whereas it could not disrupt model mammalian cell membranes at similar peptide concentrations.
2. MATERIALS AND METHODS 2.1. Materials. MSI-78 is a 22-residue synthetic R-helical peptide (amino acid sequence G-I-G-K-F-L-K-K-A-K-K-F-G-K-A-F-V-K-I-L-KK-NH2) and was synthesized and purified using the standard method.54 Different lipids were purchased from Avanti Polar Lipids (Alabaster, AL), including 1,2-dipalmitoyl-an-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), 1,2-dipalmitoyl-d62-sn-glycero-3-[phospho-rac-(1-glycerol)] (d-DPPG), 1,2-dipalmitoyl-an-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG),
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and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The molecular structures of the lipids are shown in Scheme 1. Right-angle CaF2 prisms were purchased from Altos (Trabuco Canyon, CA). The CaF2 prisms were soaked in toluene for 1 day and then sonicated in Contrex AP solution from Decon Laboratories (King of Prussia, PA) for 1 h. After that, they were cleaned in methanol for 10 min and then rinsed thoroughly with deionized water. The prisms were treated in a glow discharge plasma chamber for 4 min immediately before the deposition of lipids. The LangmuirBlodgett and LangmuirSchaefer (LB/LS) methods were used to deposit the proximal and the distal leaflets of a single lipid bilayer onto the prisms, respectively. A KSV2000 LB system and ultrapure water from a Millipore system (Millipore, Bedford, MA) were used throughout the experiments for bilayer preparation, as described previously.18 In our experiments, we used negatively charged DPPG/d-DPPG or POPG/POPG bilayers to represent bacterial cell membranes. Zwitterionic DPPC/DPPC or POPC/POPC bilayers were used to model mammalian cell membranes for comparison. The transition temperature between the gel and fluid phases for POPG or POPC lipid is 2 °C whereas that for DPPG (d-DPPG) or DPPC lipid is 41 °C. All of the experiments were carried out at room temperature (∼24 °C), at which the DPPG/d-DPPG and DPPC/DPPC bilayers are in the gel phase and the POPG/POPG and POPC/POPC bilayers are in the fluid phase. In peptidelipid bilayer interaction experiments, an appropriate amount of MSI-78 aqueous stock solution was injected into a reservoir filled with 2 mL of ultrapure water (in contact with the supported lipid bilayer) to achieve the desired peptide solution concentration. The SFG spectra were then collected after the peptidelipid bilayer interaction reached equilibrium and the SFG signal became stable (around 1 h after the injection of the peptide solution into the subphase of the lipid bilayer). A magnetic microstirrer was used to ensure a homogeneous concentration distribution of MSI-78 in the subphase below the lipid bilayer. 2.2. SFG. SFG is a second-order nonlinear optical spectroscopic technique that has submonolayer surface sensitivity. The details regarding SFG theories and measurements have been published previously2746 and will not be repeated here. The SFG setup used in this study was purchased from EKSPLA. In our experiments here, two laser beams (a 532 nm visible and a frequency-tunable infrared) are overlapped in space and time on the sample, generating a signal at the sum frequency (ωvis þ ωIR = ωsum). The pulse energies of both input beams are around 100 μJ, and the beam sizes are around 500 μm. By controlling the polarization of the input and generated signal beams, information on the interfacial molecular orientation can be obtained. All of the spectra presented in this article were collected using nearly total internal reflection geometry59 with ssp (an s-polarized output SFG signal, s-polarized input visible beam, and p-polarized input IR beam, respectively ) and ppp polarization combinations with 100 shots for each data point. For peptide amide I spectra, at least five same spectra were collected and averaged for data analysis. The AMP conformation can be deduced from the peak center of the SFG amide I signal. For an R-helical structure, the amide I signal is centered at about 1650 cm1.60 The orientation of an R-helical peptide can be determined by using SFG amide I spectra collected with ssp and ppp polarization combinations.18,61 This method has been applied to examine the membrane orientation of R-helical structures such as magainin 2,18 alamethicin,23 melittin,20 cytochrome b5,26 and G protein.25
3. RESULTS AND DISCUSSION 3.1. MSI-78 in a DPPG/d-DPPG Lipid Bilayer. After the MSI78 stock solution was injected into the subphase of the DPPG/ d-DPPG (DPPG as the proximal leaflet and d-DPPG as the distal leaflet) bilayer, the SFG signal at 1650 cm1 was monitored as a function of time. At a peptide concentration of 300 nM, no discernible SFG signal was detected in the amide I frequency 7761
dx.doi.org/10.1021/la201048t |Langmuir 2011, 27, 7760–7767
Langmuir
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Figure 2. Relation between the χppp/χssp ratio and the orientation angle of MSI-78 molecules in a DPPG/d-DPPG bilayer.
Figure 1. SFG ppp and ssp amide I spectra collected from a DPPG/dDPPG bilayer in contact with a (top) 400 nM or (bottom) 600 nM MSI78 solution.
region. This can be explained by the fact that the interaction between MSI-78 molecules and the DPPG/d-DPPG bilayer could be very weak at this low peptide concentration. For higher concentrations, the SFG signal at 1650 cm1 was detected and reached equilibrium about 1 h after the peptide injection into the subphase. SFG ppp and ssp spectra of MSI-78 in a DPPG/dDPPG bilayer were then collected. Figure 1 shows the spectra collected after a 400 (top) or 600 nM (bottom) MSI-78 solution was placed in contact with the DPPG/d-DPPG bilayer (after 1 h). In Figure 1, all amide I SFG spectra exhibit a dominant peak centered at 1650 cm1, indicating that MSI-78 adopts an Rhelical structure associated with a DPPG/d-DPPG bilayer.60 The solid lines in Figure 1 are the fitting results. Details of the SFG peak fitting methods have been described in previous publications18 and will not be reiterated here. According to the fitting results, we obtained the measured ppp and ssp signal strength ratio (or χppp/χssp) of 2.0 for 400 nM or 1.5 for 600 nM MSI-78 solution. By using this ratio, we can determine the orientation angle of MSI-78 in the DPPG/d-DPPG bilayer. SFG orientation analysis for an R-helical structure has been described in our previous publications.18,61 We defined that orientation angle θ represents the tilt angle between the principal axis of the R-helical MSI-78 molecule and the DPPG/d-DPPG bilayer surface normal. The relation between the measured χppp/χssp ratio of MSI-78 and R-helix orientation angle θ can be deduced using the developed method, assuming a δ-orientation distribution (Figure 2).61 Using Figure 2 and the measured χppp/χssp ratios, we deduced that the orientation angle θ of MSI78 associated with the DPPG/d-DPPG bilayer is around 70° (or 25°) when the bilayer is in contact with the 400 (or 600) nM MSI-78 solution. Other concentrations of MSI solutions were also tested in the experiment. As shown in Figure 2, when the MSI-78 solution concentration increased, the tilt angle θ decreases, meaning that the orientation of MSI-78 molecules changes from surface orientation to transmembrane in the
DPPG/d-DPPG bilayer. At 400 nM, MSI-78 molecules are tilted 70° from the bilayer normal. At a peptide concentration of 500 nM, the orientation angle changed from 70 to 65°. When the peptide concentration was increased to 600 nM, the orientation angle changed to 25°, which is close to the transmembrane orientation. This shows that the increase in the peptide concentration enables the MSI-78 molecules to change their orientation from perpendicular to the DPPG/d-DPPG bilayer normal to the parallel orientation. As the concentration continued to increase to 800 nM, the orientation angle of MSI-78 molecules was determined to be approximately 15° versus the bilayer normal. At an even higher concentration of 2000 nM, the measured χppp/χssp ratio equals 1.3, which is below the possible values in the calculated curve assuming a δ-orientation distribution (Figure 2). Under the assumption that every MSI-78 molecule adopts the same orientation (or a δ-orientation distribution), the value of χppp/χssp should be between 1.40 and 2.05. The observation of a low peptide concentration is in excellent agreement with previous solid-state NMR studies54 whereas the change in the tilt angle at higher concentrations (500 to 800 nM) indicates that the peptide could be aggregating to insert into the hydrophobic region of the bilayer. Such transmembrane orientations were not observed from NMR experiments;58 therefore, we believe that there are two different mechanisms in operation for low (
