Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy

Abstract

We have used high-speed atomic force microscopy to study the dynamics of bacteriorhodopsin (bR) molecules at the free interface of the crystalline phase that occurs naturally in purple membrane.Our results reveal temporal fluctuations at the crystal edges arising from the association and dissociation of bR molecules, most predominantly pre-formed trimers. Analysis of the dissociation kinetics yields an estimate of the inter-trimer single-bond energy of -0.9 kcal/mol. Rotational motion of individual bound trimers indicates that the inter-trimer bond involves W10-W12 tryptophan residues.

High-speed atomic force microscopy coming of age

“…Imaging rate, 1.03 fps (×20 playback); scan size, 800 × 800 nm 2 [98]. ・Crystallization of a CaCO 3 thin film from supersaturated solution [72] ・Ultrafast imaging of collagen [58] ・Brownian motion & photo-degradation of π-conjugated polyrotaxane [73] ・DNA translocation and looping by type III restriction enzyme [74] ・Biotinylated DNA-streptavidin interaction [75] Miles Miles Shinohara Takeyasu Trimitsu 2008 5 ・Anisotropic diffusion of point defects in streptavidin 2D crystals [76] ・Identification of intrinsically disordered regions of proteins [77] ・Human chromosomes in liquid [78] ・DNA-nuclease interaction [ ・Dynamic equilibrium at the edge of bR 2D crystals [81] ・Structural change of CaM and actin polymerization on streptavidin 2D crystals [82] ・Unidirectional translocation of cellulase along cellulose fibers [83] ・Translocation of EcoRII restriction enzyme along DNA [84] ・Fabrication and imaging of hard material surface [85] ・Purple membrane in contact-mode HS-AFM [86] ・Thermal motion of π-conjugated polymer chain [87] ・Opening of 3D hollow structure of DNA Origami [88] ・Opening of 3D hollow structure of DNA Origami [89] ・ATP-induced conformational change in P2X 4 ・Walking myosin V along an actin filament [91] ・Photo-induced structural change in bR [92] ・2D crystal formation of annexin A-V and height change of p97 [93] ・Analysis of components covering magnesotome surface [94] ・Time course of cell death by antimicrobial peptide [95] ・Dissolution of extreme UV exposed resist films under developing [96] ・Dissolution of extreme UV exposed resist films under developing [97] ・Process of forming supported planar lipid bilayer [98] ・Self assembly of amyloid-like fibrils [99] ・Effect of ClpX on FtsZ polymerization ...…”

“…Many bR-trimers diffuse in the fluidic edge regions and a small number of ATP synthase c-rings also diffuse there [144]. The dynamic association and dissociation between diffusing bR trimers and the crystalline edges were previously imaged (movie S11) and analyzed by my group and the association energy between two bR molecules was estimated to be −1.5 k B T (T = 300 K) per a single bond [81]. C-rings with 6.5 nm diameter were observed to assemble into dimers with dynamic association distances ranging from 8 to 12 nm (figure 10a).…”

“…Typical examples of this class are the studies on membrane-mediated protein-protein interaction [101] and dynamic interaction of isolated bR trimers with the edge of bR 2D crystals [81].…”

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

“…We improved the spatial resolution to capture the dynamics of membrane proteins at a high resolution and succeeded in visualizing lateral movement and structural changes of proteins in isolated biologic membranes. [17][18][19] HS-AFM is, at present, capable of imaging soft biologic molecules non-invasively without damage, suggesting that it has the potential to capture the molecular dynamics of the surface of a living cell. The bacterial cell, however, has a substantially higher topography than isolated molecules.…”

“…[14][15][16] Moreover, the dynamics of membrane proteins isolated from bacterial cells can be visualized at high resolution with HS-AFM. [17][18][19] HS-AFM will become even more useful in biologic science if it can be used to observe the molecular dynamics of living cell membranes. A large number of membrane proteins play important roles in cell function (e.g., transport, signal transduction for extracellular stimuli, and maintenance of cellular structure).…”