Dynamicsofphosph_省略_dongoldsubstrate_T_Y

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Dynamics of phospholipid membranes and nanoparticles
discerned on gold substrate
T. Yamada a*, S. Matsunaga a,b, T. Kobayashi a, M. Kawai a,b
a RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
b The University of Tokyo, Kashiwa-shi, Chiba 277-8561, Japan
*E-mail address:tayamada@riken.jp
Scanning tunneling microscopy (STM) and other surface-scientific techniques can be utilized to explore the microscopic dynamics of biological molecules in the context that the techniques are applicable for solid surfaces immersed in aqueous solutions. We devised STM, vibrational spectroscopies, and visible-light excitation techniques to make usable for molecular monolayers at solid-liquid interface. We attempted to observe dynamic motions of phospholipid layers formed on octanethiol-terminated gold (111)single-crystalline substrates placed in aqueous buffer solutions (in situ STM). By in situ STM we could observe dihexanoyl-sn-glycero-3-phosphocholine (DHPC), a relatively short kind of lipid, forming a fluidic monolayer. A crystalline phase of this monolayer was observed by applying an electrode potential compatible with the membrane potentials of real cells, and individual DHPC molecules were discerned. Furthermore, mixed lipid layers have been examined by STM. We found some nanometer-scale raft structures (phase-separated domains), which are functionally characteristic for real cell membranes. We also studied phospholipid particles suspended in buffer solutions. Suspensions were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), which are commonly found in real cell membranes. Ultrasonic homogenization in a phosphate buffer generated suspensions consisting of nanometer-scale phospholipid particles with narrow size distribution. By means of dynamic light scattering (DLS), we confirmed the suspensions from pure POPC or POPC+POPE mixtures were almost purely composed of particles with a hydrodynamic diameter of a few tens of nanometers. Hereafter we call them “minimal lipid particles (MLP)”. When the modified Au(111) substrate was immersed in these suspensions, by in situ STM, we observed adsorption of particles with a diameter ~ 10 nm forming a monolayer along the surface. It is known that some categories of antibiotics selectively attack lipids contained in germ cell membranes and disintegrate the whole cells. We chose “duramycin”, a 19-residued peptide antibiotic, which specifically binds ethanolamines. When the total concentration of phospholipid was controlled between 100 μM and 500 μM, a layer of MLP with a diameter of ~ 8 nm were discerned. During STM scanning, 7 μM of duramycin solution was added into the suspension, and the POPC+POPE MLP became fragile and seemed to be scratched by the tip, ending up with a widespread multilayer. For pure POPC MLP, duramycin caused no effect. Only a small amount of duramycin was needed to melt MLPs containing POPE. This sort of highly leveraged effect of duramycin is characteristic in the action of antibiotics. Through this experience, we realized the advantage of STM in monitoring the live nanometer-scale reactions of phospholipid entities, which have not been recognized experimentally so far. We expect more application of STM in physiological investigation in cell biology.
Keywords: STM, DLS, phospholipid membranes, nanoparticles, cell biology。