Abstract:Amphidinol 3 (AM3) is an anti-fungal polyene extracted from a marine dinoflagellate. Here, we examined the ion channel activity and membrane-embedded structure of AM3 using a lipid bilayer method and atomic force microscopy (AFM). AM3 exhibited large-conductance (~1 nS) and non-selective single-channel activity only when sterols were present in the membrane leaflet of the AM3-added side. The variable conductance suggests the formation of a multimeric barrel-stave pore. At high AM3 concentrations, giant-conduct… Show more
“…The wide range of techniques that have been used to measure AMPs activity (i.e., fluorescence resonance energy transfer (FRET), small angle neutron scattering (SANS), nuclear magnetic resonance (NMR), calorimetry, electrophysiology, X-Ray, or circular dichroism (CD)) provide little information on the oligomerization states which is an important piece of information missing in our understanding of the activity of the peptides 29 ; as oligomerization controls the nature, dynamics and regularity of the membrane deformations. Atomic force microscope (AFM) and electrochemical scanning tunnelling microscopy (EC-STM) are unique in their capability of label-free high resolution imaging of biological molecules and their surroundings under physiological conditions, yet, in the literature, few studies address the effect of AMPs on supported lipid bilayers by AFM [32][33][34][35][36][37][38][39][40][41][42][43][44] (Dap in particular was never studied), most are prior to 2011, few are the recent publications [42][43][44] . The published AFM data examines the evolution of the contours of membranes patches exposed to AMPs in solution.…”
mentioning
confidence: 99%
“…Never have the dynamics of the activity of AMPs been imaged at molecular resolution, for example oligomers could never be visualised due to their thermal diffusive motion. AFM and EC-STM molecular imaging was restricted to the static final states of the processes 32,39,42,43 , hence, overlooking the core of the AMPs molecular activity. Overall, the incapability to assert AMPs molecular activity hampers the straightforward correlation of the AFM data with the molecular, but averaged, information issued from the prevalent techniques of monitoring of AMPs molecular activity 36 .…”
The increase in speed of the high-speed atomic force microscopy (HS-AFM) compared to that of the conventional AFM made possible the first-ever visualisation at the molecular-level of the activity of an antimicrobial peptide on a membrane. We investigated the medically prescribed but poorly understood lipopeptide Daptomycin under infection-like conditions (37 °C, bacterial lipid composition and antibiotic concentrations). We confirmed so far hypothetical models: Dap oligomerization and the existence of half pores. Moreover, we detected unknown molecular mechanisms: new mechanisms to form toroidal pores or to resist Dap action, and to unprecedently quantify the energy profile of interacting oligomers. Finally, the biological and medical relevance of the findings was ensured by a multi-scale multi-nativeness—from the molecule to the cell—correlation of molecular-level information from living bacteria (Bacillus subtilis strains) to liquid-suspended vesicles and supported-membranes using electron and optical microscopies and the lipid tension probe FliptR, where we found that the cells with a healthier state of their cell wall show smaller membrane deformations.
“…The wide range of techniques that have been used to measure AMPs activity (i.e., fluorescence resonance energy transfer (FRET), small angle neutron scattering (SANS), nuclear magnetic resonance (NMR), calorimetry, electrophysiology, X-Ray, or circular dichroism (CD)) provide little information on the oligomerization states which is an important piece of information missing in our understanding of the activity of the peptides 29 ; as oligomerization controls the nature, dynamics and regularity of the membrane deformations. Atomic force microscope (AFM) and electrochemical scanning tunnelling microscopy (EC-STM) are unique in their capability of label-free high resolution imaging of biological molecules and their surroundings under physiological conditions, yet, in the literature, few studies address the effect of AMPs on supported lipid bilayers by AFM [32][33][34][35][36][37][38][39][40][41][42][43][44] (Dap in particular was never studied), most are prior to 2011, few are the recent publications [42][43][44] . The published AFM data examines the evolution of the contours of membranes patches exposed to AMPs in solution.…”
mentioning
confidence: 99%
“…Never have the dynamics of the activity of AMPs been imaged at molecular resolution, for example oligomers could never be visualised due to their thermal diffusive motion. AFM and EC-STM molecular imaging was restricted to the static final states of the processes 32,39,42,43 , hence, overlooking the core of the AMPs molecular activity. Overall, the incapability to assert AMPs molecular activity hampers the straightforward correlation of the AFM data with the molecular, but averaged, information issued from the prevalent techniques of monitoring of AMPs molecular activity 36 .…”
The increase in speed of the high-speed atomic force microscopy (HS-AFM) compared to that of the conventional AFM made possible the first-ever visualisation at the molecular-level of the activity of an antimicrobial peptide on a membrane. We investigated the medically prescribed but poorly understood lipopeptide Daptomycin under infection-like conditions (37 °C, bacterial lipid composition and antibiotic concentrations). We confirmed so far hypothetical models: Dap oligomerization and the existence of half pores. Moreover, we detected unknown molecular mechanisms: new mechanisms to form toroidal pores or to resist Dap action, and to unprecedently quantify the energy profile of interacting oligomers. Finally, the biological and medical relevance of the findings was ensured by a multi-scale multi-nativeness—from the molecule to the cell—correlation of molecular-level information from living bacteria (Bacillus subtilis strains) to liquid-suspended vesicles and supported-membranes using electron and optical microscopies and the lipid tension probe FliptR, where we found that the cells with a healthier state of their cell wall show smaller membrane deformations.
“…5 ), suggesting that AM3 more effectively enhances the order of the disordered membrane than that of ordered membrane. It is reported that AM3 forms domain-like aggregate on the membrane [ 18 ], which may increase the membrane order.…”
Section: Resultsmentioning
confidence: 99%
“…Our previous study using surface plasmon resonance (SPR) and solid-state 2 H NMR demonstrated that AM3 directly interacts with sterols in membranes [ 17 ]. In addition, more recent channel recording experiments further suggest that AM3 forms both barrel-stave and toroidal pores depending on the AM3 concentration; a higher concentration of AM3 forms jumbo toroidal pores, while a lower concentration of AM3 forms a barrel-stave channel that shows a single channel property [ 18 ]. Fig.…”
Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate
Amphidinium klebsii
, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 μM, as in the case of methyl-β-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.
“…Membrane manipulation is not limited to physical and chemical procedures conducted on the bilayer itself, but now include alterations to membrane-incorporated channels, such as channel forming substances, which may deform the membrane upon channel formation. [80][81][82] Our picture of channel-membrane interplay is more and more realistic through touching the membrane and designing more sophisticated membranes.…”
Fluidity and mosaicity are two critical features of biomembranes, by which membrane proteins function through chemical and physical interactions within a bilayer. To understand this complex and dynamic system, artificial lipid bilayer membranes have served as unprecedented tools for experimental examination, in which some aspects of biomembrane features have been extracted, and to which various methodologies have been applied. Among the lipid bilayers involving liposomes, planar lipid bilayers and nanodiscs, recent developments of lipid bilayer methods and the results of our channel studies are reviewed herein. Principles and techniques of bilayer formation are summarized, which have been extended to the current techniques, where a bilayer is formed from lipid-coated water-in-oil droplets (water-in-oil bilayer). In our newly developed method, termed the contact bubble bilayer (CBB) method, a water bubble is blown from a pipette into a bulk oil phase, and monolayer-lined bubbles are docked to form a bilayer through manipulation by pipette. An asymmetric bilayer can be readily formed, and changes in composition in one leaflet were possible. Taking advantage of the topological configuration of the CBB, such that the membrane's hydrophobic interior is contiguous with the surrounding bulk organic phase, oil-dissolved substances such as cholesterol were delivered directly to the bilayer interior to perfuse around the membrane-embedded channels (membrane perfusion), and current recordings in the single-channel allowed detection of immediate changes in the channels' response to cholesterol. Chemical and mechanical manipulation in each monolayer (monolayer technology) allows the examination of dynamic channel-membrane interplay.
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