Microsecond dynamics of a tunable-allosteric switch regulate the activity of a disaggregation machine

Large protein machines are tightly regulated through allosteric communication channels. Here we study ClpB, a protein disaggregation chaperone, whose six subunits contain a unique coiled-coil structure, the middle domain (M domain), proposed as a regulatory element that binds the co-chaperone DnaK. We study the M domain during the chaperone cycle using single-molecule FRET spectroscopy and find it to toggle on the microsecond time scale between two major states. Importantly, the ultrafast dynamics imply that ClpB’s function as an ATP-fueled disaggregating nanomachine must be controlled by the relative population of the two states, rather than their separate propensities. This mode of dynamic control enables fast adaptation to cellular needs and may be a general mechanism for the regulation of cellular machineries. To further understand the role of M domain in the allosteric communication between ClpB subunits we design our next experiments to follow simultaneously motions occurring at two adjacent subunits. To this end we will construct ClpB dimmers that hold two different FRET pairs by combining classical methods of labeling cysteine residues with the famous unnatural amino acid (UAA) technology of Schultz and co-workers to introduce a third, orthogonal labelling/linking site that can be exploit by click chemistry.