Measuring ligand–receptor binding kinetics and dynamics using k-space image correlation spectroscopy

Accurate measurements of kinetic rate constants for interacting biomolecules are crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiologi...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2014-03, Vol.66 (2), p.273-282
Main Authors: Brandão, Hugo B., Sangji, Hussain, Pandžić, Elvis, Bechstedt, Susanne, Brouhard, Gary J., Wiseman, Paul W.
Format: Article
Language:English
Subjects:
Animals
Cercopithecus aethiops
Chemical kinetics
Cholera toxin
Cholera Toxin - chemistry
COS Cells
Fluctuation spectroscopy
Fluorescence microscopy
Fluorescent Dyes - chemistry
Green Fluorescent Proteins - chemistry
Humans
Image correlation spectroscopy
Kinetics
Ligands
Microscopy, Fluorescence
Microtubule-Associated Proteins - chemistry
Microtubule-Associated Proteins - metabolism
Microtubules
Microtubules - chemistry
Microtubules - metabolism
Molecular Docking Simulation
Neuropeptides - chemistry
Neuropeptides - metabolism
Spectrometry, Fluorescence
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Summary:Accurate measurements of kinetic rate constants for interacting biomolecules are crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiological responses under different conditions. Here, we extend the k-space image correlation spectroscopy (kICS) technique to study the kinetic binding rates of systems wherein: (a) fluorescently labelled, free ligands in solution interact with unlabelled, diffusing receptors in the plasma membrane and (b) systems where labelled, diffusing receptors are allowed to bind/unbind and interconvert between two different diffusing states on the plasma membrane. We develop the necessary mathematical framework for the kICS analysis and demonstrate how to extract the relevant kinetic binding parameters of the underlying molecular system from fluorescence video-microscopy image time-series. Finally, by examining real data for two model experimental systems, we demonstrate how kICS can be a powerful tool to measure molecular transport coefficients and binding kinetics.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2013.07.042