Real time imaging of rotating molecular machines

Observation of true rotation has been relatively rare in living systems, bu t there may be many molecular machines that rotate. Molecular rotations acc ompanying function can be imaged in real time under an optical microscope b y attaching to the protein machine either a small tag such as a single fluo rophore or a tag that is huge compared with the size of the protein. As an example of the former approach, axial rotation of an actin filament sliding over myosin has been measured quantitatively by attaching a fluorophore ri gidly to the filament and imaging the orientation of the fluorophore contin uously by polarization microscopy, As a huge tag in the latter approach, an actin filament turned out to be quite useful. Using this tag, the enzyme F -1-ATPase has been shown to be a rotary stepper motor made of a single mole cule. Further, the efficiency of this ATP-fueled motor has been shown to re ach almost 100%. The two examples above demonstrate that one can now image conformational changes, which necessarily involve reorientation, in a singl e protein molecule during function. Single-molecule physiology is no longer a dream.