|Budget Amount *help
¥7,400,000 (Direct Cost : ¥7,400,000)
Fiscal Year 1996 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥5,200,000 (Direct Cost : ¥5,200,000)
A living body, which is continuous almost by definition, does not allow true rotation which requires indefinite sliding of one part against another. At the level of molecules, however, sliding is commonly encounterd, and repeated use of identical subunits leads to the formation of helical structures including rings and disks. Sliding along a helix results in rotation. Thus, biological molecules may well rotate during function. Although observation of molecular rotation has so far been scarce, except for the irregular Brownian rotation, the reason could be technical difficulties in detecting rotation of an object as small as a molecule.
In this research project, we proposed, and have realized, two approaches toward detection of molecular rotations. One is the real-time measurement of the orientation of a fluorophore through fluorescence polarization imaging. The other is to attach a huge probe to the molecule of interest and directly observe the rotation.
As an example of the former approach, we have been able to detect quantitatively the axial rotation of an actin filament sliding over myosin. For the latter, we have been able to demonstrate that a single molecule of F1-ATPase, a part of ATP synthase, is by itself a rotaty motor in which a central rod-shaped subunit rotates against surrounding subunits. We have also been able to quantify the torsional Brownian motion of a single actin filament by attaching to the filament a pair of plastic beads.
The successful imaging of molecular rotations above demonstrates that conformational changes (which necessarily involve reorientation) of a single protein molucule can now be detected during function. The techniques should help develop the new research field of single-molecule physiology.