Sensing Cooperativity in ATP Hydrolysis for Single Multisubunit Enzymes in Solution

Abstract

Cooperative interactions are critical for multisubunit enzymes to fulfill their enzymatic cycle in a coordinated fashion. To study this poorly understood process in the mammalian double-ring 16-subunit chaperonin TRiC/CCT, ATP number distributions in various hydrolyzed states are measured for single copies of the enzyme as each of the subunits can bind and hydrolyze ATP. Fluorescent-nucleotide-bound chaperonins are localized in free solution by closed-loop feedback provided by an Anti-Brownian ELectrokinetic trap (ABEL trap), producing fluorescence emission traces which allow determination of the number of nucleotides on each enzyme. As ADP molecules are dissociating from the chaperonin, the single peak at eight ADP bound simply falls in height over time, indicating a highly cooperative ADP release process difficult to observe by ensemble-averaged methods (figure).By adding AlFx during ATP incubation, ATP transition state mimics (ADP•AlFx) are locked to the complex and show a dominant peak at 8 nucleotides for all incubation concentrations above 25 μM. Although ensemble averages of the single-molecule data can be matched with standard cooperativity models, surprisingly, the observed number distributions depart significantly from standard models and reveal stronger cooperativity, illustrating the power of the single-molecule distribution-based approach.View Large Image | View Hi-Res Image | Download PowerPoint Slide