From Microstates to Macrostates in the Conformational Dynamics of GroEL: a Single-Molecule FRET Study

The chaperonin GroEL is a multi-subunit molecular machine that assists in protein folding in the E. coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. However, a measurement of subunit dynamics and their relation to the allosteric cycle of GroEL has been missing. Here, we report single-molecule FRET measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the co-chaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-liganded conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis, confirming the essential role of the observed dynamics in the function of GroEL. Significance Statement GroEL is a large protein-folding machine whose activity is accompanied by considerable conformational motions. Here, we use single-molecule FRET spectroscopy in combination with photon-by-photon statistical analysis to characterize the motions of a single GroEL subunit in real time and in the presence of ADP, ATP, and the co-chaperone GroES. Our results reveal transitions between four conformations on a timescale much faster than the functional cycle. We show that the motions of an individual subunit are directly coupled to the concerted allosteric mechanism of GroEL. This work, therefore, further demonstrates the impact of fast conformational dynamics on the biochemical function of molecular machines. ### Competing Interest Statement The authors have declared no competing interest.