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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
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Enzyme-Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism
Rohanah Hussain1, Charlotte S Hughes2, Giuliano Siligardi3
1Diamond Light Source Ltd., Chilton, Didcot, UK. rohanah.hussain@diamond.ac.uk.
Methods in Molecular Biology (Clifton, N.J.)
|November 28, 2019
Summary
Circular dichroism (CD) spectroscopy reveals protein structure and ligand interactions. This guide details CD methods, including synchrotron radiation CD (SRCD), for analyzing conformational changes and binding events.
Area of Science:
- Biophysical Chemistry
- Structural Biology
- Spectroscopy
Background:
- Circular dichroism (CD) spectroscopy is crucial for analyzing protein secondary and tertiary structures.
- It detects global conformational changes, protein folding, and local tertiary structure alterations.
- CD is particularly valuable for studying protein-ligand binding interactions.
Purpose of the Study:
- To provide a comprehensive overview of performing circular dichroism (CD) experiments.
- To detail methods, tips, and experimental designs for investigating ligand-binding interactions using CD.
- To highlight the advantages of synchrotron radiation CD (SRCD) for enhanced sample analysis.
Main Methods:
- Standard qualitative and quantitative CD measurements (single and 96-well plate modes).
- CD titrations and UV protein denaturation assays.
- Synchrotron Radiation Circular Dichroism (SRCD) using a highly collimated micro-beam.
Main Results:
- CD spectroscopy provides unique insights into ligand-induced conformational changes.
- SRCD at Diamond Light Source (DLS) enables analysis with reduced sample volumes.
- Methods for obtaining high-quality, artifact-free CD and SRCD data are described.
Conclusions:
- CD spectroscopy is an essential technique for studying protein structure and dynamics.
- The described methods facilitate robust investigations of protein-ligand interactions.
- SRCD offers significant advantages for sensitive and efficient biomolecular analysis.
