Stepping Rotation of F1-ATPase Revealed by
Single-Fluorophore Imaging


                                                                                 [ATP] = 20 nM

Fluorescence from a single fluorohpore is polarized along its long axis.  Thus, polarization measurement reveals the orientation of the fluorophore.  We attached a single fluorophore of tetramethylrhodamine to the central g subunit of F1-ATPase.  We decomposed the fluorescence from this fluorophore into the vertically (V) and horizontally (H) polarized components, and imaged the two simultaneously.  When the fluorophore is in the vertical orientation, it should show up in the V image.  Rotation into the horizontal orientation should result in the appearance of a bright spot in the H image.  The movie shows alternation of spot intensities, indicating rotation of the fluorophore.  Precise analysis revealed that the fluorophore stepped among three orientations separated by 120°.  At the end of the movie, the spots in the two images disappears suddenly, indicating that they indeed represented a single fluorophore.
                                                 Adachi, K et al.  Proc. Natl. Acad. Sci. USA. 97, 7243-7247 (2000).