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. ### Competing Interest Statement The authors have declared no competing interest.