Protein interactions with specific DNA sequences are crucial in the control of gene expression and the regulation of replication. Single-molecule methods offer excellent capabilities to unravel the mechanism and kinetics of these interactions. Here, we develop a nanopore approach where a target DNA sequence is contained in a hairpin followed by a ssDNA. This system allows DNA-protein complexes to be distinguished from bare DNA molecules as they are pulled through a single nanopore detector, providing both equilibrium and kinetic information. We show that this approach can be used to test the inhibitory effect of small molecules on complex formation and their mechanisms of action. In a proof of concept, we use DNAs with different sequence patterns to probe the ability of the nanopore to distinguish the effects of an inhibitor in a complex mixture of target DNAs and proteins. We anticipate that the use of this technology with arrays of thousands of nanopores will contribute to the development of transcription factor binding inhibitors.

No abstract

Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner.