To address how the occupancy of the serine active sites of TtClpP affects the compressibility of TtClpP, we have examined the conformational transition of the protein from its extended state to its compressed state by means of near-equilibrium pulling experiments. Toward this end, a steered MD protocol was used, pulling at constant velocity a stiff spring of force constant equal to 100 kcal/mol Å. The transition coordinate was defined as the Euclidian distance separating the center of mass of the upper hemisphere to that of the lower hemisphere computed from the position of all α-carbon atoms. The length of the transition coordinate was determined on the basis of apo-TtClpP and the structure of the compressed conformation (PDB: 3QWD), and amounts to 8 Å. Three independent, 200-ns-long realizations were performed for the empty protein and three trajectories for ClpP containing either two (in one trajectory) or three (in the other trajectories) zwitterionic alanine tripeptides, pulling irreversibly the two lobes of the protein from an extended state to a compressed one.

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