Jul 29, 2025
Isothermal titration calorimetry protocol for binding studies of enterovirus EV-A71 2A protease and small inhibitors
- Eda Capkin1,2,3
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Eda Capkin 2025. Isothermal titration calorimetry protocol for binding studies of enterovirus EV-A71 2A protease and small inhibitors. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyqroqvx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 23, 2025
Last Modified: July 29, 2025
Protocol Integer ID: 218847
Keywords: Isothermal titration calorimetry, ITC, EV-71 2A protease, protein-ligand binding, thermodynamics, dissociation constant, drug discovery, binding affinity, calorimetry, protease inhibitors, 2a protease from enterovirus stratin, isothermal titration calorimetry protocol, isothermal titration calorimetry, studies of enterovirus ev, enterovirus stratin, enterovirus ev, binding study, a71 2a protease, ligand concentration, small inhibitors this protocol, small molecule inhibitor, small inhibitor, drug discovery application, binding interaction, protein
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This protocol describes an isothermal titration calorimetry (ITC) method for measuring binding interactions between the 2A protease from enterovirus stratin EV-71 and small molecule inhibitors.
Experiments are performed at 25°C using 25 μM protein and 250 μM ligand concentrations with 17 sequential injections. Data analysis determines binding parameters (KD, stoichiometry) for drug discovery application.
Materials
Materials:
HEPES:HEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
NaCl: Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
TCEP: 0.5 M TCEP: Pierce™ Bond-Breaker® TCEP SolutionThermo ScientificCatalog #77720
DMSO: Pierce™ Dimethylsulfoxide (DMSO), Sequencing GradeThermo ScientificCatalog #10127403
PCR tubes for sample:BrandTech™ BRAND™ Thin Wall 0.2 mL PCR TFisher ScientificCatalog #13-882-58
Buffer: 50 mM HEPES pH 8.0, 150 mM NaCl, 0.5 mM TCEP
Protocol materials
HEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
0.5 M TCEP: Pierce™ Bond-Breaker® TCEP SolutionThermo ScientificCatalog #77720
Pierce™ Dimethylsulfoxide (DMSO), Sequencing GradeThermo ScientificCatalog #10127403
BrandTech™ BRAND™ Thin Wall 0.2 mL PCR TFisher ScientificCatalog #13-882-58
Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632
Troubleshooting
Safety warnings
Warning: The buffer solution in protein and compound (detergent%, DMSO%) should be the same to minimize mismatch and dilution.
The concentration of samples should be known and correct.
Protein expression and purification
Thre protocol used for expression and purification is the one below using the following plasmid Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632
Protocol
CREATED BY
Korvus Wang
Buffer preparation
Stock 5X Buffer: 250mM HEPES pH 8.0, 750 mM NaCl, 2.5 mM TCEP
Prepare 100 mL of 1X buffer by diluting the 5X stock solution with Milli-Q water.
Remove 2 mL from the 1X buffer, then add 2 mL of 100% DMSO to make a 2% DMSO buffer.
Protein preparation
Prepare 25 µM protein solution with buffer including 2% DMSO at 400 µL.
The PEAQ-ITC instrument has a cell volume of 300 µL but you can prepare at 400 µL in any case and not to inject air.
Compound preparation
100 mM stocks in 100% DMSO
Take 0.2 μL and add 9.8 μL running buffer without DMSO. This will make a running buffer in 2% DMSO. Then, add 70 uL 2% DMSO + running buffer.
Final concentration: 250 micromolar (µM)
Run settings
As a start without any affinity information, 20 -30 μM protein and 200 -300 μM sample concentrations are suggested.
Cell (protein) 25 µM at 400 µL
Syringe (samples) 250 µM at 80 µL
Temperature: 25˚C
No of injections: 17
Reference power:10 µcal/s
Initial delay: 60 seconds
1st injection 0.8 μL, rest 2.4 μL
Spacing: 150 seconds
Stirring Speed (RPM): 750
Feedback Mode: High
Instrument preparation and run
The syringe is usually left in the rest position and detached from the adaptor. Attach the syringe to the fill port adaptor and place it for the cleaning part. For the cell, put the cleaning part into the cell.
In the ITC Control software, select Wash for both cell and syringe, and start the procedure
There are two Hamilton syringes, one is used for water and buffer wash/injections, and the other is used only for protein not to dilute protein with buffer solution
After the cleaning, manually wash 4-5 times with water, then perform 4 – 5 times manual injections with buffer to equilibrate the cell.
After the wash, manually inject protein solution into the cell till you can see from the top (300 μL).
In the software, check the syringe position. If it is not ready to “sample load”, click the ‘plunger down’.
Place the sample containing the eppendorf in the holder and insert the fill port adaptor attached syringe.
Then, select the ‘sample load’ to fill the syringe with sample.
Detach the fill adaptor and place the syringe into the cell.
Enter the sample and protein concentration for the syringe and cell sections in the software, respectively.
Start the run
Results expected and data analysis
Data Analysis
Open the ITC Analysis Software and upload the result.
Check the overall heat change plot over the 17 injections.
Data evaluation and adjustment for an ITC data
Check the baseline and adjust it if it is noisy.
Baseline adjustment in the raw titration plot
Assign control as blank or fitted offset to subtract the blank response.
Then, check the KD, N values, and other parameters.
Adjusted titration plot and binding isotherm with thermodynamic parameters
After the completion of the run, start the cleaning process as stated in steps 28 to prepare for next run.
Protocol references
- ITC200: CMI Getting Started Guide to Isothermal Titration Calorimetry ( https://cmi.hms.harvard.edu/sites/g/files/omnuum4016/files/2025-03/CMI%20ITC200%20Getting%20Started%20Guide.pdf)
- Manual: MicroCal PEAQ-ITC user manual (https://www.malvernpanalytical.com/en/learn/knowledge-center/user-manuals/man0573en)