Analysis of Linked Equilibria.

JiaBei Lin, Aaron L Lucius

Methods in enzymology 2015

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The ATPases associated with diverse cellular activities (AAA+) is a large superfamily of proteins involved in a broad array of biological processes. Many members of this family require nucleotide binding to assemble into their final active hexameric form. We have been studying two example members, Escherichia coli ClpA and ClpB. These two enzymes are active as hexameric rings that both require nucleotide binding for assembly. Our studies have shown that they both reside in a monomer, dimer, tetramer, and hexamer equilibrium, and this equilibrium is thermodynamically linked to nucleotide binding. Moreover, we are finding that the kinetics of the assembly reaction are very different for the two enzymes. Here, we present our strategy for determining the self-association constants in the absence of nucleotide to set the stage for the analysis of nucleotide binding from other experimental approaches including analytical ultracentrifugation. © 2015 Elsevier Inc. All rights reserved.