Bayesian Inference of Binding Kinetics from Fluorescence Time Series.
J Shepard Bryan1,2, Stanimir Asenov Tashev3,4, Mohamadreza Fazel1,2
1Department of Physics, Arizona State University, Tempe, Arizona 85281, United States.
The Journal of Physical Chemistry. B
|May 7, 2025
Summary
This study introduces a new method to accurately measure binding and unbinding rates from fluorescence data, even with noise and photobleaching. The approach reliably infers kinetic rates alongside photobleaching, improving binding kinetics analysis.
Area of Science:
- Biophysics
- Biochemistry
- Molecular Dynamics
Background:
- Analyzing binding kinetics using fluorescence time traces is challenging due to measurement noise and photophysics.
- Photobleaching, unlike photoblinking, cannot be easily mitigated and limits current methods for determining binding and unbinding rates.
Purpose of the Study:
- To develop a novel method for inferring binding and unbinding rates alongside photobleaching rates from fluorescence intensity traces.
- To overcome limitations of existing methods caused by noise and photobleaching in kinetic rate analysis.
Main Methods:
- A two-stage process utilizing a hidden Markov model (HMM) to analyze individual regions of interest (ROIs).
- Inferring fluorescence intensity levels and state trajectories from each trace using the HMM.
- Employing the inferred state trajectories from all ROIs to determine kinetic rates.
Main Results:
- The proposed method effectively analyzes noisy fluorescence traces.
- It accurately accounts for photobleaching events during kinetic analysis.
- Provides reliable uncertainties for the inferred binding kinetics.
Conclusions:
- The developed method offers a robust approach to studying binding kinetics in the presence of noise and photobleaching.
- Demonstrated effectiveness through simulations and DNA origami binding experiments.
- Enhances the accuracy and reliability of fluorescence-based kinetic measurements.
Related Concept Videos
01:19Protein Dynamics in Living Cells
2.0K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
2.0K
02:18The Equilibrium Binding Constant and Binding Strength
12.7K
The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
12.7K