Global fitting analysis of a protein-ligand binding experiment

A few weeks ago AFFINImeter launched an Isothermal Titration data Analysis challenge of the analysis of a protein-ligand binding experiments. The participant had to globally analyze a set of Isothermal Titration Calorimetry experiments using AFFINImeter and get the thermodynamic and structural parameters of the interaction between both molecules (the receptor protein and the ligand).

The participants in this contest had the opportunity to demonstrate their ability to propose the right model for a given binding isotherm as well as to get the corresponding parameters upon fitting using AFFINImeter. On their side, less experienced participants had the opportunity to:

• Learn the difference between stoichiometric and site equilibrium constants.
• Perform a global analysis of several isotherms
• How to find the, eventually unknown, concentration of active protein in a given experiment

The results recently published by Henlz et al in [Methods 59 (2013) 336-348] were taken as a reference to generate the set of ITC isotherms selected for this contest.

The Winner of the ITC data analysis contest:

The winner of the contest is Professor Deborah Yablonski. She provided the most accurate and reliable analysis amongst all the participants in our challenge.

 Professor Deborah Yablonski
Head of The Immune Cell Signaling Lab
Rappaport Faculty of Medicine
Technion – Israel Institute of Technology

 

Deborah Yablonski is a biochemist and molecular biologist, specialized in the study of Immune Cell Signaling Pathways. Her research has illuminated the molecular mechanisms by which adaptor proteins, and their associated protein kinases,  regulate T cell responsiveness to antigen.

Debbie’s comments after participating in the contest:

“I enjoyed participating in the contest, as it was a very helpful way to learn how to use the AFFINImeter program. We are now attempting to use the program to analyze our own data.”

The Solution of the contest

The data proposed for the contest will remain available here, in case you want to learn more about it or to try it by yourself. The material prepared to guide the participants through the two steps are also available in our blog. But if you want to go directly to see the solution just keep reading and watch the following Video  Tutorials.

Experimental Conditions

A VP-ITC instrument with a cell volume of 1.4103 mL was chosen to simulate all the curves at 298 K using AFFINImeter. Several tips were provided by our scientific team during the contest period, suggesting that the analysis could be performed in two steps of different complexity:

STEP 1: Analysis of a single Protein-Ligand binding experiment

It consists of a standard protein-ligand binding ITC experiment where a ligand solution, initially located in the syringe, is titrated into the sample cell containing a protein solution.

The solution is not straightforward since the protein is able to simultaneously bind up to two ligand molecules. Another important aspect of the experiment is that the concentration of the active protein is not accurately known (this is not uncommon in an actual experiment). In this case, the active protein concentration is known to have an uncertainty of 10%. Thus, the parameter rM (available in AFFINImeter just to correct for the concentration of the compound in the sample cell) is also expected to be fitted, in addition to the equilibrium constants and the enthalpies of the thermodynamic processes. Since no ligand dilution, blank experiment was performed the Qdil parameter (the dilution molar enthalpy of the injected compound) should also be fitted. Finally, two different approaches are available in AFFINImeter to analyze these data, namely “stoichiometric equilibrium” and “independent sites” approaches. It is instructive to perform the analysis in both ways to compare the results.

STEP 2: Analysis of a Competitive Protein-Ligand Binding experiment

Four different binding isotherms involving one or two ligands able to simultaneously bind the protein are expected to be globally fitted in this step. This can be easily done in AFFINImeter by linking parameters (equilibrium constants and enthalpy changes describing the same binding event) between different curves.

 

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