From Microstates to Macrostates in the Conformational Dynamics of GroEL: A Single-Molecule Förster Resonance Energy Transfer Study.

The chaperonin GroEL is a multisubunit molecular machine that assists in protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. Here, we report single-molecule Förster resonance energy transfer 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 cochaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-ligated conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis. These coinciding results from bulk measurements for an entire ring and single-molecule measurements for a single subunit provide new evidence for the concerted allosteric transition of all seven subunits.

Designed active-site library reveals thousands of functional GFP variants.

Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is sensitive to mutations due to a high density of molecular interactions, substantially reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called high-throughput Functional Libraries (htFuncLib), that designs a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We apply htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recover >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be used in one-shot optimization of activity in enzymes, binders, and other proteins.