Real time imaging of rotating molecular machines

Abstract

Observation of true rotation has been relatively rare in living systems, but there may be many molecular machines that rotate. Molecular rotations accompanying function can be imaged in real time under an optical microscope by attaching to the protein machine either a small tag such as a single fluorophore 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 rigidly to the filament and imaging the orientation of the fluorophore continuously 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 F₁-ATPase has been shown to be a rotary stepper motor made of a single molecule. Further, the efficiency of this ATP-fueled motor has been shown to reach almost 100%. The two examples above demonstrate that one can now image conformational changes, which necessarily involve reorientation, in a single protein molecule during function. Single-molecule physiology is no longer a dream.—Kinosita, Jr., K. Real time imaging of rotating molecular machines.