Visualization of mobility by atomic force microscopy

Intrinsically disordered regions (IDRs) of proteins are very thin and hence hard to be visualized by electron microscopy. Thus far, only high-speed atomic force microscopy (HS-AFM) can visualize them. The molecular movies identify the alignment of IDRs and ordered regions in an intrinsically disordered protein (IDP) and show undulation motion of the IDRs. The visualized tail-like structures contain the information of mechanical properties of the IDRs. Here, we describe methods of HS-AFM visualization of IDPs and methods of analyzing the obtained images to characterize IDRs. © 2012 Springer Science+Business Media New York

Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. Based on these simulation results, we speculate that the conformational change of the hinge domain might be essential for the dsDNA binding regulation and play roles in condensin-mediated DNA-loop extrusion

Active damping of the scanner for high-speed atomic force microscopy

金沢大学大学院自然科学研究科物理学金沢大学理学部The scanner that moves the sample stage in three dimensions is a crucial device that limits the imaging rate of atomic force microscopy. This limitation derives mainly from the resonant vibrations of the scanner in the z direction (the most frequent scanning direction). Resonance originates in the scanner\u27s mechanical structure as well as in the z piezoactuator itself. We previously demonstrated that the resonance originating in the structure can be minimized by a counterbalancing method. Here we report that the latter resonance from the actuator can be eliminated by an active damping method, with the result the bandwidth of the z scanner nearly reaches the first resonant frequency (150 kHz) of the z piezoactuator. © 2005 American Institute of Physics

高速原子間力顕微鏡の開発

取得学位：博士(理学)，学位授与番号：博甲第754号，学位授与年月日：平成17年9月30日,学位授与年：200

High-resolution imaging of myosin motor in action by a high-speed atomic force microscope

金沢大学理学部The atomic force microscope (AFM) is a powerful tool for imaging biological molecules on a substrate, in solution. However, there is no effective time axis with AFM; commercially available AFMs require minutes to capture an image, but many interesting biological processes occur at much higher rate. Hence, what we can observe using the AFM is limited to stationary molecules, or those moving very slowly. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behaviour of biomolecules. For this purpose, we have developed various devices optimised for high-speed scanning. Combining these devices has produced an AFM that can capture a 100 x 100 pixel image within 80 ms, thus generating a movie consisting of many successive images of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica, in solution

Tip-sample distance control using photothermal actuation of a small cantilever for high-speed atomic force microscopy

金沢大学 理工研究域 数物科学系We have applied photothermal bending of a cantilever induced by an intensity-modulated infrared laser to control the tip-surface distance in atomic force microscopy. The slow response of the photothermal expansion effect is eliminated by inverse transfer function compensation. By regulating the laser power and regulating the cantilever deflection, the tip-sample distance is controlled; this enables much faster imaging than that in the conventional piezoactuator-based z scanners because of the considerably higher resonant frequency of small cantilevers. Using this control together with other devices optimized for high-speed scanning, video-rate imaging of protein molecules in liquids is achieved. © 2007 American Institute of Physics