Membrane Orientation of MSI-78 Measured by Sum Frequency Generation Vibrational Spectroscopy

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. In order to better understand the role of AMP-lipid interactions on the functional properties of AMPs, we determined the membrane orientation of an AMP (MSI-78 or pexiganan) in various model membranes using sum frequency generation (SFG) vibrational spectroscopy. 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 model bacterial cell membrane. Supported 1,2-Dipalmitoyl-an-Glycero-3-Phosphocholine (DPPC) bilayer or 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) bilayer was used as 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. On the other hand, when the concentration was increased to 600 nM, MSI-78 molecules change orientation to make a 25° tilt from the lipid bilayer normal; whereas multiple orientations were observed for even higher peptide concentration in agreement with toroidal-type pore formation as reported from 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 (~12000 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 lamellar phase POPG bilayer and also found to be inducing toroidal-type pore formation. The deduced lipid flip-flop from SFG signals observed from lipids also supports MSI-78 induced toroidal-type pore formation.

A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells

“…39 The biophysical studies show that both peptides interact preferentially with the bacterial cell membrane model, the membrane disruption being concentration-dependent. 40 The proposed interaction of both peptides with the bacterial membrane initiates with peptide binding to the membrane surface through electrostatic interactions, which is especially pronounced in the case of MSI-78. In the case of MSI-78(4−20), its higher hydrophobicity (higher H and μH) can favor a deeper insertion within the bacterial lipid bilayer, which may explain its ability to exert a bacterial effect despite its lower ability to form α-helices.…”

Combining Total Internal Reflection Sum Frequency Spectroscopy Spectral Imaging and Confocal Fluorescence Microscopy

“…12 Subsequently, controlling the polarization and frequency of the SFS excitation beams enables interfacial orientation information to be obtained. 7 In recent years, SFS has been employed for contrast generation in imaging applications. SFS microscopy as a stand-alone technique is both label-free and nondestructive and has been used for investigations in both material 5,17 and biological 18,19 systems.…”

Molecular Insights in the Structure and Layered Assembly of Polyelectrolytes at the Oil/Water Interface

“…By implementing different polarization combinations of the incident and detected beams, different orientational components of the interfacial molecules can be probed. This technique has already been a useful tool for studying charged biological macromolecules at air-water, [39][40][41][42][43][44][45][46] solid-liquid, 39,41,44,45,[47][48][49][50][51][52][53][54] and membrane interfaces; 39,[44][45][46][55][56][57][58][59][60] including, polyelectrolytes at the air-water 61-63 and solid-liquid [64][65][66][67][68][69] interfaces. While informative, these previous VSF studies do not capture the role of a unique oil-water interfacial environment on polyelectrolyte adsorption and assembly.…”