Global fitting: the key for a robust analysis

Download use case: Global Fitting

 

The Indian parable of “The six blind men and the elephant” tells the story of six blind men who touch an elephant in the hope of learning what it is like. As each one can only feel a different part of the animal the individual conclusions obtained are in disagreement and none of them provides a real view of the full elephant. “only by sharing what each of you knows can you possibly reach a true understanding”; that´s the moral behind this nice story.


 

Fig 1: The six blind men and the elephant: only a global analysis of the overall data provides a true understanding.

The binding assay(s) achieved to characterize a molecular interaction often provides not just one, but several binding curves from which the affinity constant is obtained.
Sometimes, an individual fit of these curves yield a set of binding constants that are significantly different from them; this result can be very confusing because, in principle, these binding curves are a representation of the same binding event and should converge to provide the analogous information. Often, the explanation for this behaviour is that the different curves indeed provide only partial and/or different information of the interaction, not enough to unequivocally determine the binding affinity through individual analysis.


“It´s like feeling only a separate part of the elephant”


This is a typical scenario when facing the study of complex binding events that involve more than one equilibrium and several binding curves are obtained, i.e. from different frequencies of the spectra in a titration experiment, from data registered using different techniques (ITC, NMR, Optical Spectroscopies…) and/or from experiments performed at different concentrations of the species participating in the binding event.


Analogous to the parable of the six men and the elephant, the way to get a true understanding of the binding event consist of the global analysis of the different curves.

Fig 2: The binding curve obtained from 2D NMR titrations.


Being aware of the relevance of global analysis, in AFFINImeter we count with the possibility to perform Global fitting of multiple data to tailored binding models where one or more fitting parameters are shared between isotherms. The number and identity of the parameters shared are selected by the user.
Moreover, two or more parameters can be related through mathematical relationships designed by the user. All these features make our global fitting tool the most potent among others to perform a robust analysis of binding data of complex interactions.

About the disuses of  Isothermal Titration Calorimetry in drug discovery research

Isothermal Titration Calorimetry (ITC) is the gold standard for the calculation of affinity in molecular interactions. Many times, researchers claim that the high consumption of sample does not offset the use of ITC for Kd calculation.
Conversely, ITC hides many surprises in the acquisition data that can provide more information in a single experiment that other techniques that are more expensive and more complicated to use.

Download the PDF file of Implementation of kinITC into AFFINImeter

 

1. ITC collects data from the interaction as a function of time that can be analyzed to obtain kinetic information (kon and koff values). It can cover a very similar range as Surface Plasmon Resonance in a “label-free” and “in-solution” manner (Fig 1).

2. ITC can also provide valuable information about the mechanism of interaction. The high sensitivity of the ITC sensor makes it sensitive to more intriguing interactions as conformational changes, cooperativity…

Using a global fitting approach for the analysis of the isotherms and a model builder to create tailored binding models, the different mechanisms of interaction can be confirmed and characterized.

Find attached a couple of publications describing the application of this new method for ITC data analysis:

Download the PDF file of Implementation of kinITC into AFFINImeter

 

The concepts of stoichiometric and site binding constants

Download: The concepts of stoichiometric and site binding constants

The interaction between a monovalent ligand L and a multivalent receptor R involves the presence of various species, including the complex of R fully saturated with a number of ligands, and intermediate complexes of R partially saturated. This scenario can be described in terms of reaction schemes following two approaches:

 

  1. Based on equilibria between existing stoichiometric species (Stoichiometric approach).
  2. Based on equilibria between L and specific interaction sites of R (independent sites approach).
For a better understanding, let´s consider a particular case where L binds to a bivalent receptor:

1. Stoichiometric approach

This approach uses reaction schemes based on equilibria between stoichiometric species and yields stoichiometric binding constants. A model based on stoichiometric equilibria is valid to fit data of both independent and non-independent events and therefore, it is of wider applicability.

Here, the reaction scheme includes a first equilibrium between the free species and the intermediate RL and the second equilibrium between RL + L and RL2 (Fig. 1). The corresponding binding constants, K1 and K2, are denominated stoichiometric binding constants since they refer to equilibria between stoichiometric species.


2. Independent site approach

This approach uses a reaction scheme based on the binding of the ligand to individual sites present in the receptor and considering that all the sites are independent; thus, it supplies site binding constants.

In this case, the reaction scheme considers the presence of two sites in the bivalent receptor and two intermediate complexes (R, L and RL) formed when the ligand binds to s1 or s2 and consequently, the existence of a total of 4 equilibria (Fig. 2). The corresponding binding constants, Ks1, Ks2, Ks1s2 and Ks2s1, are denominated site binding constants since they refer to equilibria between L each specific site of R.


If you want to know more about how to get the stoichiometry (number of sites) and site binding constants with the independent sites approach you can click on the following button:
 

 

AFFINImeter binding models for Nuclear Magnetic Resonance

AFFINImeter is already well known in ITC binding data analysis for providing the possibility to use tailored binding models created by the user. The models are generated with the tool “model builder” that includes a letter code “M-A-B” to describe titrate (M), titrant (A) and if necessary, the presence of a third species (B) (Figure 1).

 

Fig.1 Example of a competitive binding model created in AFFINImeter where the titrant in syringe “A” binds to the titrate in cell “M” to form a 1:1 complex “MA” and a second ligand “B” mixed in the cell with “M” forms the complex “MB” and thus competes with “A”.

 

Following the same approach, the binding models available for the software AFFINImeter for Nuclear Magnetic Resonance are generated with the model builder and based on the “MAB” code. But there are significant differences between ITC and NMR data analysis when the time comes to select a binding model from AFFINImeter, which have an origin in the inherent characteristics of each technique and in the different experimental design. In chemical shift perturbation (CSP) NMR titration experiments, the observed parameter used to monitor the progress of the binding event is the chemical shift of titrate resonance signals. Hence, the models used for NMR data analysis require the presence of compound “M” (titrate) as it is the species from which changes associated with the binding process are monitored. Conversely, in ITC the observed parameter is the heat change upon interaction and this parameter is not necessarily linked to a particular species “M”, “A” or “B”.

An illustrative example is the evaluation of a monomer-dimer self-association process using NMR or ITC. In NMR, the standard experimental setup would consist in the incremental dilution of the compound sample at high concentration in the NMR tube, to monitor dimer dissociation (Figure 2a). In ITC the standard experimental setup would consist in a titration of the compound sample at high concentration in the syringe (species “A” according to the AFFINImeter code) into the calorimetric cell filled up with solvent (Figure 2b).

 

Fig.2 Representation of experimental setup for a) NMR dilution experiment and b) ITC dilution experiments. The corresponding schemes of AFFINImeter binding models for data analysis are shown.

 

 

Would you like to know more about AFFINImeter for Nuclear Magnetic Resonance? Press the button below:

AFFINImeter-NMR

 

MicroCal User´s day in Madrid

The next April 19th will be taking place in Madrid the MicroCal user´s day. The event will be held at the Institute of Chemical Physics Rocasolano.

The basics and applications of the ITC -Isothermal titration Calorimetry and DSC -Differential scanning calorimetry technologies will be treated. It will be an excellent event with very interesting talks and nice place for catch up of these applications.

We are pleased to announce that Dr Juan Sabín (Co-founder & Product Designer) will be giving a presentation about AFFINImeter, the software for Isothermal Titration Calorimetry data analysis.

Additionally, Dr Raúl Pacheco application specialist from Malvern Instruments, Dr Margarita Menendez from the Institute of Chemical Physics Rocasolano and Dr Javier Murciano from the University of Granada will also attend the event.

The Microcal user´s day has been organised by Malvern Instruments, CSIC-Spanish National Research Council, Institute of Chemical Physics Rocasolano and Iesmat.

For more information:

Microcal user´s day Madrid.

5 Tips to optimize your ITC experiments for kinetic analysis.

Since the method KinITC was implemented in AFFINImeter many researchers have been using it to obtain kinetic information of binding interactions from ITC data; the good news is that no special experimental setup different from the standard ITC experiment is required to register data for kinetic analysis! The information is derived from analysis of the thermogram of regular ITC titrations and therefore one can obtain kinetic information from old ITC data right away.

There are few recommendations though if you are planning to perform new ITC experiments, focused on getting high-quality data for kinetic analysis:

1) Set the time between successive power measurements to 1s or 2s. This will give a better definition of the thermogram peaks and therefore a more precise calculation of the equilibration times.

2) Set the time recording the baseline before the first injection to 1 or 2 minutes. In order to have a good reference when determining the signal baseline.

3) Leave enough time between injections so that a full equilibration for the overall set of injections is registered.

4) Clean thoroughly the instrument before the experiment. This is fundamental to optimize the response time of the instrument, which strongly determines the sensitivity of the kinetic analysis.

5) A high gain feedback mode is recommended in order provide the fastest response time (but, be careful because a high feedback mode can also generate signal overshooting after injection, which greatly difficulties the kinetic analysis! If overshooting happens, don´t use high gain model).

Need more information about this subject? Contact the Scientific team of AFFINImeter at info@affinimeter.com.

Follow these simple tips to increase the quality of your ITC data for kinetic analysis

Figure Junio2016

KinITC for TA and MicroCal Calorimeters – New version Release!

During the last months we’ve contacted you asking your opinion and experience with the software. Thanks to all your suggestions and comments we have improved the previous version of the software to make it easier, faster and more versatile.

What’s new in AFFINImeter?

  • Availability of KinITC for TA and Microcal data files.
  • Inclusion of Multi Temperature Analysis: Van’t Hoff and Eyring plots
  • The project management section has improved, now you can easily organize your projects in Folders/Subfolders and move them from one to another.

 

Easier, Faster and more Versatile!

In this new version you will find several changes adressed to improve the user experience of the software, adding more features, making it easier and faster to user, and more versatile.

 

Go to the software!

Request an AFFINImeter Online Demo

AFFINImeter is a software designed to further exploit the potential of your Isothermal Titration Calorimetry instrument.

Contact us to request an Online Demo

We want to offer our help to guide you through AFFINImeter. If you are interested in the analysis of a particular ITC data and you don’t know which binding model you should apply or how to design it with AFFINImeter, do not hesitate to contact us!

affinimeter-free-online-demo

 

Meanwhile you can consult our educational material (Videotutorial, Cases of Use, Notes and Webinars) in our Web Page or read the Tutorials and examples section.

Remember that as a launching promotion you can use AFFINImeter free during the first 6 months!

ITC data analysis contest – New submission Date

A few days ago AFFINImeter launched an Isothermal Titration Calorimetry (ITC) data analysis contest. At this point no one has been able to finish the challenge, eventhough there is a considerable amount of participants. Since the main goal of the contest is to give all our AFFINImeter users an educational tool to get experience in the process of ITC data analysis.

Therefore, we would not be satisfied if some of you ran  out of time. For this, we have decided to extend the contest deadline to January 31st

Join the Challenge

We encourage all people interested in molecular binding analysis, not only based on ITC data but also on any other experimentaltechnique, to participate in this challenge. If you are an expert in the analysis of thermodynamic data this is a nice opportunity for you!

Find the best combination of model & parameters for the target data and win a Kindle Fire HD 7 just sending us your results.

tablet kindle fire HD7
Isothermal Titration Calorimetry Data Analysis Contest

If you are a beginner in the analysis of ITC or, in general, of binding experiments, this challenge is also perfect for you since it will give you the opportunity to learn how to analyse actual ITC measurements. Our scientific team has selected some really interesting experiments and prepared some instructions that will guide you in the analysis process.

Learn the difference between stoichiometric and site equilibrium constants

By trying to solve this contest you will learn the difference between stoichiometric and site equilibrium constants, you will see how to easily perform a global analysis of several isotherms, and even how to find the, eventually unknown, concentration of active protein in a given experiment. Sign up HERE  and you will get free access to AFFINImeter. Please, do not hesitate to encourage your labmates to join us. People from the whole world are already participating!

Download Poster here
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