Multiple Independent Sites: Advanced tools for a successful analysis.

Isothermal titration Calorimetry experiments of a ligand binding to a macromolecule with multiple independent sites

A successful Isothermal Titration Calorimetry (ITC) experiment requires the acquisition of high quality experimental data together with a careful analysis. Choosing the right binding model to fit the ITC isotherm is critical in order to get the true thermodynamic profile of the interaction. Often, the main limitation to achieve good results arises when the evaluation software lacks of the mathematical model that best describes our binding experiment. A good example is the case of a ligand binding to a macromolecule with multiple independent sites, i.e ligand – DNA interactions (1). Until now the readily available mathematical models to fit such experiments was limited to one or two sets of “n” independent identical sites; frequently, these models offer a poor description of the interaction due to the inherent higher complexity of the system, where many distinct binding equilibria coexist.

 

Multiple Sets of Independent Sites

AFFINImeter ITC offers an unlimited number of user-defined binding models. Particularly, it counts with a feature to easily design models based on multiple independent binding sites. Here, a model with a number of sets of independent sites can be created with no limitation in the number of sets or sites. Noteworthy, the number of sites in each set can be considered as a fitting parameter throughout the data analysis. As an illustration, the following figure shows the reaction parameters of a model generated with AFFINImeter that describes a ligand binding to a receptor having 3 sets of sites, each set having an unknown number of sites. Fitting the experimental data to such model yields the microscopic association constant (K) and the change in enthalpy (ΔH) of the ligand binding to each site type, and the number of sites in each set (n).

Scheme of the interaction of a ligand with a multisite receptor
Reaction parameters table of a model that describes the interaction of a ligand (in syringe) with a multi-site receptor (in cell) having 3 sets of independent sites. Note that the option “Fit” was checked to consider the number of sites (n) as fitting parameters.

These binding models, described by numerous variable parameters, may end up in an over-parameterized fitting function. Thus, the best strategy to achieve a robust and consistent analysis involves the global fitting of several ITC curves acquired under different experimental conditions. In this sense, AFFINImeter also supports global fitting of multiple isotherms wherein parameter linkage between curves is used to decrease the relative number of estimated parameters per experiment.

References:
(1) Methods 2007, 42, 162–172.

Use of precise standard reactions for Isothermal Titration Calorimeter validation

Use of  precise Standard Reactions for Isothermal Titration  Calorimeter Validation

Many published papers report inconsistent thermodynamic values of the same interactions between chemical reactants or macromolecular binding. One of the reasons for this discrepancies is the difficulty of repeating the same conditions in the ITC experiments (buffer, pH, concentrations, ionic strength, source of the materials…). But users start to be more aware that some systematic errors of the calorimeters may also have an important effect in the reported values.

For instance, the interaction between 4-carboxybenzenesulfonamide and bovine carbonic anhydrase II is considered a standard reaction to be measured by ITC and its enthalpy has been measured by 14 operator using different calorimeters (1). The resulting value considering all these independent measurements is -10.4±2.5 kcal·mol-1. The error of the enthalpy is surprisingly high and significantly higher than those typically reported for ITC measurements.

Baranauskiené and co-workers (1) suggest the use of precise standard reactions for  Isothermal Titration calorimeter validation after the calibration. The table below shows the series of chemical reaction they propose as standards where the enthalpy of binding has been determined to high precision and the reagents are readily available from commercial sources.

 

table-chemical-reactions-calorimeters-reference

They also used these standards reactions to compare the results obtained with different micro calorimeter. Their study concluded that Microcal calorimeters are more reliable than TA Calorimeters; and the most recent Microcal ITC200 is less accurate than Microcal VP-ITC. Nano ITC-III calorimeter results were very reproducible, but enthalpy values were systematically underestimated. To learn more about Isothermal Titration Calorimeter validation,, visit the references from where this article was taken.

VP-ITC-NanoITC-ITC200-calorimeter-comparison-entalphie
Enthalpy of the interaction between Tris base and HNO3 as a function of temperature and measured in different ITC calorimeter. Literature values were taken from (2).

References:

(1) Int. J. Mol. Sci 2009, 10, 2752-2762.

(2) Handbook of proton Ionization Heats Wiley-Interscience: Hoboken, NJ, USA, 1979

How to design an Isothermal Titration Calorimetry experiment?

Isothermal Titration Calorimetry Experiment Simulation

 

 theoretical-isotherm-Microcal-VP-ITCspecies-concentration-isothermal-titration-calorimetry-experiment

The Simulator tool available in AFFINImeter is completely free under registration. This is currently the only alternative to design complex Isothermal Titration Calorimetry (ITC) experiments. The Simulator allows plotting ITC curves (evolved heat as a function of the system concentration) together with a phase diagram of the different chemical species that are present in the solution regardless the complexity of the interaction mechanism between the involved molecules.

Scheme of a ITC Experiment
Isothermal Titration Calorimetry Experiment

 

Avoid Trial and Error Assays

Using the AFFINImeter Simulator you will be able to pre-visualize the results of an experiment, provided that you have an approach for the interaction mechanism of your molecules and of the corresponding thermodynamical parameters. This tool will guide you in the optimization of the most advantageous combination of experimental parameters: the concentration and location (in the sample cell or in the syringe) of your compounds, the injection volume and the number of titrations; thus avoiding trial-and-error assays and saving time, reactants and money.

This tool is also useful to set the conditions under which the distribution of chemical species meet some special requirement (for instance, the solution dominated by a given chemical species). It can also be used for didactic purposes since it helps to illustrate how a chemical species can be displaced by another, to explain the difference between cooperative and non-cooperative processes or to explain the effect of endothermic and exothermic processes.

 

Applications in Drug Discovery

Isothermal Titration Calorimetry is a key technique in the development of drugs since it assess the affinity between molecules. The most typical application is to determine the free energy of interaction between proteins and inhibitors. The AFFINImeter simulator tool allows simulating the displacement of a weak ligand by a strong ligand as a function of the concentration of the compounds involved in the experiment.

Advantages of the Simulator

Introduce your personalized thermodynamic model directly in chemical language (reaction scheme) and an estimation for the corresponding thermodynamic parameters. Even the most advanced models are easy to implement. Through the model builder AFFINImeter offers an unlimited amount of thermodynamic models for Isothermal Titration Calorimetry data analysis. If the model required for your system is not available, please, do not hesitate to contact us and we will try to implement it.

Sequential Binding Sites Model
This is a Sequential Binding Model that considers the free species of both solutes M and A, plus the hybrid complexes with stoichiometries (1:1, 1:2, 2:1, 2:2).

 

Start Using the Simulator

The AFFINImeter Simulator is free under registration. To learn how to use it, please read this tutorial.

The Model Builder is a versatile tool to translate binding interactions into mathematical models

The Model Builder is one of the novel features of AFFINImeter.  Through the model builder AFFINImeter offers an unlimited amount of thermodynamic models for ITC data analysis. The overall binding equilibria within the species involved in the experiments is easily drawn by the user directly in chemical language. Then AFFINImeter translates the resulting reaction scheme into robust  binding  models to be used to isotherm ITC simulation or to perform Isothermal Titration Calorimetry curve fitting.

The model builder is a versatile tool, it allows to design models involving up to three different species (i.e. the case of two ligands that compete with each other to bind a macromolecule) and has the advantage to selectively place them in the  syringe cell and/or in the calorimetric cell. 

Binding Reaction Scheme of a competitive interaction
Reaction Scheme of a Competitive Binding Interaction

It also allows the design of models for dissociation, ranging from simple homodimers to higher-order oligomers. During the model construction no mathematical equations are required, once the whole set of binding interactions is defined by the user in the reaction builder, AFFINImeter internally generates the system of equations that define the reaction scheme proposed. The new model (reaction scheme and equations) is saved  internally by AFFINImeter and listed in the user’s database so that can be utilized anytime so simulate or fit data.

AFFINImeter Reaction Builder
Competitive binding model builded with the AFFINImeter tool

AFFINImeter-ITC offers an exclusive unlimited amount of personalized model families including

  • Unrestricted Competitive Sequential Binding with no limitation in the stoichiometry of the binding model.
  • Competitive Multiple and Independent Sets of Identical and Independent Sites.
  • Dissociation of any chemical species including homogeneous n-mers, heterogeneous complexes and even micelles.

With this extensive offer of model families the user will be able to perform the thermodynamic characterization of a vast variety of biological and physicochemical processes from ITC measurements. A few examples of classical and new applications of ITC experiments that you can analyze with AFFINImeter are:

  • Protein-ligand or host-guest complex formation with unlimited stoichiometries
  • Competition of different molecules to occupy a given binding site even for high order complexes
  • Binding between ligands and polymers or large macromolecules with any number of (independent) sets and/or sites
  • Dissociation/aggregation of supramolecular heterogeneous species including protein oligomers
  • Structural and thermodynamic information of large aggregates, including micelles: aggregation number, enthalpy of formation, Gibbs energy and dilution heat of monomers and aggregates
AFFINImeter logo

AFFINImeter Beta Version Release

AFFINImeter is the most complete software for Isothermal Titration Calorimetry data Analysis. You can easily  build your own Binding Models or Perform Global Simultaneous Analysis of several Isotherms, among other features.

Here is the list of main implementations and changes in the released Beta version of AFFINImeter:

  1. A new model family, for Isothermal Titration Calorimetry Data Analysis, that considers the independent binding of one or two (competitive) different compounds to a macromolecule has been implemented. This is a generalization of the so called “two sets of independent sites (TSIS)” models that may consider any number of different and independent sets with any number of identical and independent sites per set.
  2.  Binary origin files (with OPJ extension) coming from microcal ITCs can now be directly uploaded. This facilitates the work by avoiding intermediate files and potential errors in the format. TA experiments can also be uploaded as in the previous version.
  3.  Simulation and Fitting results can be easily shared by e-mail. AFFINImeter is the only software able to make simulations for any model. This tool is free for any registered user. Simulations are extremely useful in several situations: (i) to optimize the experimental setup of an experiment; (ii) once a system has been analyzed it allows predicting the conditions under which the distribution of chemical species meet some special requirement (for instance, the solution dominated by a given chemical species); and (iii) for didactic purposes, to illustrate how a chemical species can be displaced by another, to explain the difference between cooperative and non-cooperative processes or to explain the effect of endothermic and exothermic processes
  4. Several optimizations, including calculations and also the protection of data, have been performed. Some occasional noise was detected in the calculated fitting curve of the previous version of AFFINImeter. This is connected to the noise of the experimental data points and also to the approach employed to correct for the displacement of volume in Microcal and TA ITC instruments. A new algorithm was implemented to globaly fit all the data points, thus minimizing this kind of problems
  5. It is possible to remove data points when editing dataseries as well as when uploading them to a project. This is really useful to make several tests of the same dataseries by removing different points on the experimental curve.

Functionalities of the previous version of AFFINImeter are still active, with AFFINImeter you can:

  • Design your own sequential binding model by using our model builder tool. It is possible to design models, from an Isothermal Titration Calorimetry experiment, involving up to three different compounds with two ligands that compete with each other to bind a macromolecule. The second ligand may be in the cell or/and in the syringe. Dissociation of any order (from simple homodimers to complex hetero-oligomers) induced by dilution experiments can also be easily analyzed.
  • Analyze the presence of local minima by repeating fittings starting from different random seeds for the parameters
  • Include dynamic relationships between parameters
  • Include dynamic bounds to restrain the fitting parameters
  •  Correct for the effective concentration of any of the compounds of the experiment by fitting a scaling factor
  • Fit several curves simultaneously to minimize the statistical uncertainty of the fitting parameters
  • Manage associate accounts for your students and collaborators. Administrator account owners will have access to the full activity of their associated users
  •  Share your results by e-mail with your collaborators. They will get direct access to the measurements, fitting curve and fitted parameters of the experiments you decide to share with your colleagues.
  • Organize your projects and access them from any device

Calorimetry Conference, CALCON 2014 (II)

The second day at CALCON 2014 started with Matthew Auton (Mayo Clinic of Minnesota) receiving the “Sting Sunner Memorial Award”. He presented his work on the thermodynamics properties of the domains of the von Willebrand factor, which mutations results in the so-called von Willebrand disease, the most common inherent bleeding disorder in man.

During the plenary session, Heiko Heerklotz (University of Toronto) showed interesting results of ITC experiments titrating surfactant into lipidic liposomes and lipidic solution into micelles systems. Analysis of this complex isotherms provides information to characterize the phase boundaries between micellar phase, fluid, ordered and gel-phase membranes.

Calorimetry Conference poster
The Calorimetry Conference, CALCON 2014

Joseph Emerson (Mississippi State University) presented his work in complex equilibria associated with metal ions coordination in dioxygen-activating enzymes. His group uses ITC data to understand how this enzymes discriminate between available metal ions.
The “hot topic” in the other thematic sessions and during the posters presentation continues being the study of structure and stability of DNA quadruplexes related to the human telomere sequence.
AFFINImeter presented the poster “AFFINImeter: A new tool to analyze Isothermal Titration Calorimetry experiments” where we showed how AFFINImeter opens up new possibilities for the application of the ITC technique such as:

AFFINImeter Applications to Isothermal Titration Calorimetry and other spectroscopy Techniques
Examples of new applications that can be studied with AFFINImeter

 

 

Calorimetry Conference, CALCON 2014 (I)

The 69th Calorimetry Conference (CALCON 2014) hold in Santa Fe (New Mexico, USA) opened today with Peter R. Tremaine (University of Guelph) receiving the “Hugh M. Huffman Memorial Award”. In his opening lecture he reviewed his research on modeling the behaviour of aqueous systems near the critical point; where many geological and industrial processes take place: from nuclear reactors to the origin of life.
In the plenary session Thomas Record (University of Wisconsin) discussed the effect of the Hofmeister series salts in the interaction with biopolymers and Randy Black (Quantum Design Inc) presented a new calorimetry cell for their measurement system to measure heats exchanges at mK temperature.
Micheael Henzl (University of Misouri) opened the “Macromolecule-Ligand Interaction” session presenting the study of the effect of mutations of Parvalbumin on its interaction with different ions. Parvalbumin is a vertebrate-specific protein believed to function primarily as cytosolic Ca2+ buffers. He showed global analysis of different ITC isotherms using a competitive binding model for Ca2+ and Mg2+ , when both ions are loaded in the syringe of the ITC equipment.

CALCON 2014
Calorimetry Conference, CALCON 2014

Cocaine-binding DNA aptamers have been developed for use in biosensor applications as they can be selected to bind almost any target with high specificity and affinity. Philip Johnson (York University) showed ITC competitive experiments between cocaine and quinine to explain the mechanism of binding with this DNA apatmers.
During the “Acid Folding and Interactions” session, different students from the Lewis Biophysics Laboratory (Mississippi State University) presented their works on the interaction of G-quadruplex DNA, one of the promising anti-cancer targets, with different ligands. They showed complex ITC isotherms with two inflexion points which they analysed with a binding model that takes into account three different events: the binding of the first ligand, the unfolding of the G-quadruplex DNA structure and the binding of a second ligand.
Tomorrow AFFINImeter will be in the poster session presenting the poster “AFFINImeter: A new tool to analyze Isothermal Titration Calorimetry experiments”. On Wednesday we will present an oral communication on the same topic during the “Global Analysis, Modeling and Simulations” session.

“Molecular Recognition: new tools for complex situations”

June 24, 2014 Spanish National Research Council (CSIC)

Conference póster Eva Muñoz
“Molecular Recognition: new tools for complex situations”

Dr. Eva Muñoz, a member of our scientific department, was invited to the Centre for Organic Chemistry Manuel Lora Tamayo (CSIC) in Madrid where she gave a talk entitled “Molecular Recognition: new tools for complex situations”. During the talk she described various examples of complex interacting systems difficult to evaluate with standard analytical methods, and the use of new experimental and analytical approaches to circumvent these problems. Here, AFFINImeter was presented as a novel analytical tool for the analysis of complex ITC data.
Moreover, she introduced AFFINImeter in a seminar at the Biological Research Centre, CIB (CSIC, Madrid) explaining how it can be a must tool in the molecular recognition research field. She described the main features of the application like the “model builder”, an easy-to-use tool with which researchers design their own binding models, the “global fitting” of a series of isotherms or the analytical tool to explore possible “local minima” generated from the fitting procedure.

CALCON 2014, 69th Calorimetry Conference

Dr. Juan Sabin, a member of our scientific department, will participate with an oral presentation at the 69th Calorimetry Conference (Santa Fe, NM 2014) to describe the main features of AFFINImeter like the “model builder”, an easy-to-use tool with which researchers will design their own binding models, the “global fitting” of a series of isotherms or the analytical tool to explore possible “local minima” generated from the fitting procedure.

He will show how AFFINImeter opens up new possibilities for the application of the ITC technique such as:

  • Thermodynamic Characterization of ligand induced conformational changes.
  • Competitive binding models with high order complexes.
  • Quantitative analysis discrimination of ligands in heterogeneous mixtures (i.e. isomers, heteropolymers…).
  • Accurate analysis of ultra low-affinity or ultra high affinity complexes.
  • Characterization of binding processes with no restriction in the stoichiometry.
  • Micellization studies to obtain the aggregation number, enthalpy and Gibbs energies of micelle formation.
  • Global simultaneous analysis of isotherms (i.e. global analysis of curves from direct and reverse experiments).

Don’t miss the talk at http://www.calorimetry-conference.org /

 

20th International Symposium on Surfactants in Solution (Coimbra, Portugal)

The AFFINImeter innovation team has developed an original model to analyze dissociation ITC isotherms of aggregates ranging from (protein) dimers to large supramolecular clusters (like micelles).  With this model the average number of molecules in the aggregates, the molar free energy and enthalpy of transfer from the aggregate to the solution and the dilution of both monomers and aggregates can be obtained. This model soon will be available in the AFFINImeter Software.

The validation of this model comparing our results with those determined from other experimental methods is going to be presented in the 20th International Symposium on Surfactants in Solution (SIS, June 2014), where our CSO Angel Piñeiro is giving Oral presentation on Monday  June 23, 2014.