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. Author manuscript; available in PMC: 2013 Jan 10.
Published in final edited form as: Nat Struct Mol Biol. 2007 Sep 16;14(10):921–926. doi: 10.1038/nsmb1300

Figure 5.

Figure 5

Absence of nucleotide exchange after ParM polymerization. Fluorescent etheno-ATP (εATP) was used to induce polymerization in the nonhydrolyzing ParM mutant E148A. Acrylamide was used as a collisional quencher to increase the signal from the bound nucleotide and to monitor protection of the nucleotide from interaction with solvent. Adding ParM (5 μM) to εATP caused a rapid jump in fluorescence, consistent with monomer binding, followed by a slower increase, whose time course is identical to that of polymerization. When prepolymerized ParM (10 μM) was added to εATP, there was no jump in fluorescence. When a 100-fold excess of unlabeled ATP was added to the polymerized ParM, only a very slow decrease in fluorescence (with a half-life on the order of 1 h) was seen that was not consistent with rapid nucleotide exchange on the filament. This time course is orders of magnitude slower than exchange on monomeric ParM, and the kinetics are consistent with turnover of subunits in the filament.