Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

DIRECT OBSERVATION OF TRANSLOCATION BY A AAA+ MACHINE Gilad Haran 1 ; Remi Casier 1 ; Inbal Riven 1 ; 1 Weizmann Institute of Science, Rehovot, Israel The mechanism by which AAA+ machines translocate substrate proteins through their lumen remains a subject of contention. Structural studies suggest the ‘hand-over-hand’ mechanism, by which subunits move from one side of the machine to the other, while hydrolyzing ATP and pulling the substrate along the way. However, biochemical and biophysical experiments are in dispute with this model. Here, we utilize single-molecule FRET spectroscopy to directly probe translocation by the AAA+ machine ClpB. We isolate ClpB and substrate protein molecules within surface-tethered lipid vesicles and allow them to interact repeatedly. Surprisingly, the observed translocation events, although dependent on ATP, occur in only milliseconds, much faster than ATP hydrolysis. Further, the translocation rate depends only weakly on temperature and ATP concentration. Using three-color FRET experiments, we observe both forward and backward substrate motion, though most events take place in the ‘correct’, forward direction. Replacing ATP with the slowly hydrolyzable analog ATP γ S abolishes both rapid translocation and directionality. Finally, measurements of the motion of pore loops, ClpB’s structural elements that interact with substrates and pull them in, reveal microsecond up-and-down motions. Combined together, these findings indicate a fast, stochastic Brownian-motor-like mechanism, redefining how ATP is coupled with mechanical action in AAA+ machines.

38