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.  

How to perform a neat Isothermal Titration Calorimetry experiment?

In a Isothermal Titration Calorimetry experiment, If the injected volume or any of the concentrations is too small, or if the ratio between both concentration values is not appropriate, then the signal-to-noise ratio will be low and the uncertainty of any result will be high.

Scheme of a ITC Experiment
Isothermal Titration Calorimetry Experiment

Determinant factors of poor quality results in Isothermal Titration Calorimetry Experiments

The control of these factors might be limited by the available amount or the corresponding solutes (they can be expensive or difficult to synthesize/purify). If the total number of titrations is low then the solute in the sample cell will not be saturated and the quality of the results will be poor. Additionally, in reactions for which multiple chemical species can be formed it is always better to simultaneously fit several experimental data series, each focusing the sampling in a different concentration region which is more sensitive to any of the species.

Advantages of prior Isotherm Simulation

The simulator tool provided by AFFINImeter allows you to test the effect of the parameters listed above on your experiment in order to optimize its design, thus saving time and samples.

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Reliability of the thermodynamic parameters obtained from Isothermal Titration Calorimetry experiments

How to obtain thermodynamic parameters?

 

Binding Reaction Scheme
Protein-Ligand Binding Scheme

Isothermal Titration Calorimeters have been designed to determine the thermodynamic parameters corresponding to physicochemical processes that take place in a solution: protein-ligand binding, enzyme assay, drug encapsulation, dissociation/aggregation, micellization, etcThe reliability of the thermodynamic parameters obtained from Isothermal titration calorimetric experiments depends critically on:

(i) the volume injected in the sample cell upon each titration

(ii) the concentration of the solution in the sample cell

(iii) the concentration of the solution in the injection system

(iv) the number of titrations

(v) the number and relative concentration of the different chemical species that can be formed upon mixing of both solutions

(vi) the heat involved in the formation of those chemical species

(vii) the order in which the solutions are considered, i.e., which solution is introduced into the sample cell and which is in the injection system.

 

Global Fitting Analysis in ITC Displacement Titrations with AFFINImeter

ITC displacement titrations offer an attractive alternative to standard assays when working with ultra high- or ultra low- affinity interacting systems. The method requires the fitting of at least two isotherms that share various adjustable parameters. AFFINImeter counts with advanced tools, like the global fitting of multiple dataseries and the analysis of isotherms registered under unusual experimental design, which can facilitate the analysis and expand the range of applications of isothermal titration calorimetry experiments. As an illustration, herein we present a displacement titration assay to determine the thermodynamics of HIV-protease with indinavir, a high affinity binder, and with acetyl-pepstatin, a weaker ligand. Using AFFINImeter a global analysis of four isotherms was performed describing: HIV-protease binding to indinavir (I) or to acetyl-pepstatin (II): HIV-protease binding to indinavir incorporating acetyl-pepstatin in the cell (III) or in the syringe (IV).

Isothermal Titration Calorimetry is one of the most commonly used approaches to obtain affinity and thermodynamic data of molecular interactions and has become a routine method in the pharmaceutical industry.1 Isothermal titration Calorimetry is applicable to numerous interacting systems, as long as a detectable heat change is produced during complexation, covering an important range of binding affinities (106 ≤ KA ≤108 M-1). Nevertheless, standard ITC experiments present some limitations in the case of very low- or very high-affinity interactions (i.e. affinities in the low millimolar or high nanomolar range, respectively). High affinity interactions (KA ≥ 109 M-1) yield square-shaped isotherms whose fitting yield accurate values of the binding enthalpy but only estimates of the association constant. Attempts to recover a sigmoidal shape requires the use of very low concentrations of the interactants that, in most cases, is not feasible in the practice (the minimum concentration that will typically cause a confidently measurable heat change for a 1:1 interaction is about 10 μM). On the opposite, low affinity interactions should be studied at high concentrations and this requirements is often a serious limiting step due to various potential reasons like limited solubility and/or availability of the sample molecule, or the existence of aggregation processes at the required concentration. In both high- and low affinity systems these experimental drawbacks can be circumvented by using the ITC displacement method.2,3 Here, the receptor is titrated with a high affinity ligand, but in the presence of a weaker ligand in the sample cell that competes for the complexation with the receptor (figure 1). With this experimental set up the apparent affinity of the strong ligand is “artificially” lowered, obtaining a sigmoidal isotherm that yields more accurate binding data. When the goal is to obtain the thermodynamic parameters of an ultra highaffinity system, a titration with the weaker binder is performed first to obtain the corresponding affinity constant and enthalpy (KA-weak and H-weak). These values are required for the analysis of the ITC displacement experiment, where a competitive binding model is used to estimate the thermodynamic parameters of the tight binding (KA-tight and H-tight). Analogously, when the goal is to obtain information of an ultra low- affinity system a direct ITC titration of the receptor alone with a ligand of higher affinity is performed. The resulting KA-tight and H-tight are then incorporated in the analysis of the isotherm from the ITC displacement assay.

This case study exemplifies the potential advantages of using AFFINImeter in ITC displacement assays. The software offers unique advanced tools that enhance the robustness of the method and makes it more versatile, facilitating the acquisition of reliable thermodynamic data from ultra-high of ultra-low affinity systems. Thus, it opens a door for new applications of the displacement assay.
1 G. Holdgate, S. Geschwindner, A. Breeze, G. Davies, N. Colclough, D. Temesi, L. Ward, Biophysical Methods in Drug Discovery from Small Molecule to Pharmaceutical. Protein-Ligand Interactions. In Methods in Molecular Biology 2013, 1008, pp 327-355.
2 A. Vellazquez-Campoy and E. Freire, Isothermal titration calorimetry to determine association constants for highaffinity ligands. Nature protocols 2006, 1, pp 186-191.
3 W. B. Turnbull, Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Life Sciences protocol.

 

Download the case of use here

Isothermal Titration Calorimetry
Global Analysis in ITC Displacement Titrations with AFFINImeter