2P107 Curvature of Bump Structures on Purple Membrane Depending on pH and Ionic Strength Analyzed by Atomic Force Microscopy(03. Membrane proteins,Poster)

Abstract

Surface Enhance Infrared Absorption (SEIRAS) have unique properties that enhances signals at vicinity (in an order of several nm's) of a substrate metal. This property is useful to determine solely the surface chemical process distinguished from that occurred in bulk phase. Therefore when a biological sample of interest is confined to such surface, one can selectively monitor a chemical reaction of the target regardless of the complex ensemble biological process in the bulk phase. We present an application of SEIRAS to in-situ investigation for a folding process of membrane protein (bacteriorhodopsin) during cell free expression on the nano-disc artificial lipid bilayer. 2P104等温滴定型熱量計による多剤輸送担体 EmrE の基質結合様 EmrE is a substrate/proton antiporter, which extrudes a wide range of lipophilic cations. Here, we had demonstrated with ITC how tetraphenyl phosphonium ion (TPP + ) binds to the wild-type and mutants (E14Q, D, E25Q). As pH increase, the driving force of EmrE/TPP + binding shifted from enthalpy-driven to entropy-driven and the binding affinity was more potent, inferring that EmrE has a more open conformation at acidic pH, and a closed conformation at neutral and alkaline pH. Especially, Glu25 might be one of determinant residues to drive the substrate binding, because in E25Q, at pH 7.4, the contribution of enthalpy to interaction energy remained a little, but significantly. The mechanism of proton coupled substrate transport will be discussed here. 式の解析 2P105多剤排出トランスポーター The multidrug transporter AcrB actively exports a wide variety of noxious compounds using the proton-motive force as an energy source in Gramnegative bacteria. AcrB adopts an asymmetric structure comprising three protomers with different conformations that are sequentially converted during drug export. These cyclic conformational changes for drug export are called the functional rotation. The Motion Tree is a new method to describe the structural change between two states of proteins in a hierarchical manner. This method can detect the rigid body motions and clarify the relations among them. In the present study, we applied the Motion Tree analysis to the cyclic conformational changes of AcrB. As a result, the key motions for the functional rotation were observed. Purple membrane (PM) is a membrane patch which is formed by self-assembly of membrane protein bacteriorhodopsin with archaeal lipids. Atomic force microscopy (AFM) studies for PM supported onto mica substrate shows dome-like "bump" structures on the surface. Since PM must have a curvature to form the bump structure, bump formations will be related to a mechanism for structural formations via self-assembly. To elucidate effects of asymmetric distribution of charged residues between two aqueous domains on bump curvature, AFM observations were carried out toward identical PM sheets with varying solvent pH and ionic strength. The bump curvature studies indicated large influence of electrostatic interaction. The model for the bump structure will be discussed. 2P106 ABC トランスポーターにおける薬剤結合の影響...