From Microstates to Macrostates in the Conformational Dynamics of GroEL: A Single-Molecule Fo''rster Resonance Energy Transfer Study

The chaperonin GroELis a multisubunit molecular machinethat assistsin protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allostericconformational changes during its reaction cycle. Here, we reportsingle-molecule Fo''rster resonance energy transfer measurementsthat directly probe the conformational transitions of one subunitwithin GroEL and its single-ring variant under equilibrium conditions.We find that four microstates span the conformational manifold ofthe protein and interconvert on the submillisecond time scale. A uniqueset of relative populations of these microstates, termed a macrostate,is obtained by varying solution conditions, e.g., adding differentnucleotides or the cochaperone GroES. Strikingly, ATP titration studiesdemonstrate that the partition between the apo and ATP-ligated conformationalmacrostates traces a sigmoidal response with a Hill coefficient similarto that obtained in bulk experiments of ATP hydrolysis. These coincidingresults from bulk measurements for an entire ring and single-moleculemeasurements for a single subunit provide new evidence for the concertedallosteric transition of all seven subunits.