Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution

Abstract

In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperoninbound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.single molecule | allostery | fluorescence | enzymology | nucleotide counting Y ears of study of cooperativity for proteins with multiple ligand binding sites have yielded important insights, ever since the role of cooperative oxygen binding to hemoglobin in high oxygendelivery throughput was recognized (1-3). Multisubunit enzymes usually hydrolyze ATP in a concerted fashion, but actually observing this process, enzyme by enzyme, can provide a deeper picture of the underlying cooperativity resulting from communication among the various subunits. A well known example has been the study of the rotary motor F1-ATPase by single-molecule techniques (4). Another class of multisubunit cellular machines is the double-ring type I and type II chaperonins which can have from 14 to 18 subunits each of which can hydrolyze ATP (5, 6). Ensemble studies of the bacterial type I GroEL/GroES system, for example, have indicated that ATP binds subunits in one 7-membered ring with positive cooperativity, while negative cooperativity operates between rings (7). We focus on the cooperative process operative in the mammalian type II chaperonin TRiC/CCT, which has two ring-shaped cavities with built-in lids composed of eight different subunits each. TRiC is essential for the folding of a number of key proteins in mammalian cells, including actin, tubulin, and many cell cycle regulators (8). Previous ensemble measurements of ATP-induced allosteric transition rates and steady-state ATPase rate in TRiC show evidence for positive and negative cooperativity du...

The Mechanism and Function of Group II Chaperonins

“…Single molecule experiments have been performed where fluorescently labeled ATP bound to individual complexes were counted by recording discrete photobleaching steps [77]. The observed distributions centered on 8 bound nucleotides under both cycling and non-cycling conditions, meaning that half of the total nucleotide binding sites are occupied, consistent with the finding that only four subunits per ring bind ATP.…”

Single-molecule spectroscopy and imaging over the decades

“…10b), providing new insights into the mechanism of catalysis. In yet another experiment 197 , the number of ADP molecules (each labeled with a fluorophore) bound to a chaperonin enzyme TRiC can be directly counted by enumerating the number of photobleaching steps for a single enzyme in the trap (Fig. 10c).…”

“…Adapted with permission from Ref. 197. (d) Correlated intensity-lifetime-spectral photodynamics of a single Atto647N fluorophore in solution.…”

Mechanisms of Cellular Proteostasis: Insights from Single-Molecule Approaches

“…Many chaperones, such as Hsp90 and the cylindrical chaperonins, are structurally and conformationally complex, making mechanistic studies challenging. Electrokinetic trapping (45) and single-molecule fluorescence approaches (67, 77, 78) have proven particularly useful in understanding the rich dynamic behavior of some molecular chaperones.…”