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Simultaneous ligand binding to intact and partially formed ATP-binding sites in the hexameric termination factor Rho
Tyler D Billings1, Kristie Baker1, Philip Lacey1
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, USA.
The Journal of Biological Chemistry
|October 9, 2025
Summary
Native mass spectrometry reveals how ATP binds to the Rho termination factor, a hexameric helicase. This method quantifies binding events in complex systems, offering insights into macromolecular machine function.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Macromolecular machines use thermodynamic coupling to coordinate functions.
- Oligomeric systems present challenges for quantifying ligand binding.
- Structural heterogeneity can complicate biophysical measurements.
Purpose of the Study:
- To quantify ATP binding to the hexameric helicase, Escherichia coli Rho termination factor.
- To investigate ligand binding in complex macromolecular machines.
- To provide mechanistic insights into Rho function.
Main Methods:
- Native mass spectrometry was employed to measure ATP binding.
- Binding events were quantified for hexameric and lower-order Rho complexes.
- Superstoichiometric binding was observed and analyzed.
Main Results:
- ATP binding to the hexameric Rho termination factor was successfully measured.
- Quantification of ATP binding to various Rho complexes was achieved.
- Superstoichiometric binding indicated ATP interaction with partially formed sites.
Conclusions:
- Native mass spectrometry is suitable for studying ligand binding in complex oligomeric systems.
- Detailed insights into ATP binding mechanisms of Rho were obtained.
- Understanding ligand binding is crucial for elucidating macromolecular machine coordination.
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