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Updated: May 29, 2026
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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Published on: February 18, 2014
Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution
Yan Jiang1, Nicholai R Douglas, Nicholas R Conley
1Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Summary
Single-molecule studies reveal highly cooperative ATP release in the TRiC/CCT chaperonin. This cooperativity, particularly ADP release, is crucial for enzyme function and challenges standard models.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Multisubunit enzymes rely on cooperative interactions for coordinated function, but the underlying mechanisms are not fully understood.
- The TRiC/CCT chaperonin, a double-ring multisubunit enzyme, plays a vital role in protein folding.
- Understanding the dynamics of nucleotide hydrolysis and release is key to elucidating enzyme cooperativity.
Purpose of the Study:
- To investigate the cooperative ATP hydrolysis and ADP release mechanisms in single TRiC/CCT chaperonin complexes.
- To characterize the nucleotide number distributions during enzymatic cycling using single-molecule techniques.
- To compare single-molecule observations with standard cooperativity models.
Main Methods:
- Utilized single-molecule fluorescence from chaperonin-bound nucleotides.
- Employed an Anti-Brownian Electrokinetic (ABEL) trap for precise measurements in free solution.
- Observed ADP release dynamics and ATP hydrolysis transition state mimics (ADP·AlFx).
Main Results:
- Single-molecule observations revealed a highly cooperative ADP release process, with a dominant peak at 8 ADP molecules.
- ATP hydrolysis transition state mimics (ADP·AlFx) stabilized the complex with 8 bound nucleotides.
- Observed nucleotide number distributions deviated from standard cooperativity models, highlighting the need for refined mechanistic understanding.
Conclusions:
- Single-molecule analysis provides critical insights into the cooperative mechanisms of multisubunit enzymes like TRiC/CCT.
- The data suggest subunit-occupancy-dependent cooperativity plays a significant role in ATP hydrolysis.
- These findings underscore the limitations of ensemble-averaged methods for studying complex enzymatic processes.
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