Control of thermal fluctuation of proteins by evanessent field trapping.

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The aims of this study were development of new techniques to control thermal fluctuation of proteins and to apply these techniques to actin-myosin system toward the final goal that is to understand roles of thermal fluctuation in chemi-mechano couppling of actin-myosin system. During the period of this grant, we obtained the following achievments to the final goal.(1)Single molecule imaging of fluorescently-labelled proteins on metal by surface plasmons in aqueous solution : We made an optical microscope using surface plasmon resonance at the meniscus between thin metal layer and water. By using this technique, evanessent field was enhanced 2-10 times compered to that by total internal reflection between glass and water. Active movement of single molecules of the fluorescently labelled motor proteins was observed on the surface of gold and alminium.(2)Development of a technique that allows mechanical and ligand-binding events in a single myosin molecules to be monitored simultaneously … More : Using this technique, we found that the force generation of single myosin molecule does not always coincide with the release of bound nucleotide, presumably ADP. Instead the myosin head produces force several handreds of milliseconds after ADP is released.(3)Development of a new instrument to capture and manipulate individual myosin molecules using a scanning probe : We found that single myosin head moves along an actin filament with regular steps of 。ォ5.5nm. Groups of two to five rapid steps in succession often produce displacement of 11 to 30nm. This multiple stepping is produced during just one biochemical cycle of ATP.(4)Measurement of the torsional diffusion of actinfilaments : A single actin filament with bead attached to both ends was suspended in solution by optical tweezers. Torsional diffusion of the filament was observed by rotational movements of a bead in the optical tweezers in the presence of myosin with or without ATP. Amplitude of the rotation was increased in the presence of ATP, though torsional regidity of the filament was not changed. Less