Pursuing a reliable calculation of binding affinity.

The relevance of uncertainties.

 

The reliable determination of binding constants (Ka) and enthalpies (ΔH) from Isothermal Titration Calorimetry depends, among other aspects, on the quality of the thermogram recorded where a high signal-to-noise ratio is pursued to get a trusty isotherm. Low signal-to-noise ratio conducts to less precise integration of the thermogram peaks and, consequently, to a binding isotherm defined by points with larger related uncertainties. Logically, the uncertainty associated to the integral points will significantly affect the determination of the thermodynamic parameters and should not be set aside through the data analysis.

Here´s an illustrative example of two ITC experiments of the interaction between Carbonic Anhydrase II (CA) and 4-carboxybenzenesulfonamide (4-CBS) that yielded thermograms of different quality (Fig 1*).

Fig.1 ITC titrations of 4-CBS into CA. a) Exp1, higher signal-to-noise ratio; b) Exp2, lower signal-to-noise ratio.

Data fitting to a 1:1 binding model was performed for both experiments and for comparison purposes the analyses were conducted twice, taking into account the uncertainties associated to the calculation of the peaks integral (weighted fitting) and not considering them (all the points having the same weight).
The results show a larger discrepancy in the Ka values obtained from Exp2, depending on whether or not the uncertainties are included in the fitting. Besides, the Ka obtained from the weighted fitting of Exp2 is significantly closer to the corresponding Ka value of the weighted fitting of Exp1 (Figure 2).
Fig.2 KA, DH and rM (correction parameter for CA concentration in the cell) values from fitting of Exp1 and Exp2 a) without uncertainties and b) with uncertainties. The bar diagrams show the comparison between KA values of Exp1 and Exp2.

 

The main origin of this difference is the large uncertainty associated to the mid-titration point, which consideration has an effect on the final Ka value (Figure 3).
Fig.3 Fitted isotherm from Exp 2. Redline: theoretical curve calculated when considering uncertainties; black line: theoretical curve calculated with no uncertainties.

 

Being aware of the relevance of weighting data points based on their uncertainty AFFINImeter includes, into the raw data processing step, the automatic determination of uncertainties in the peaks integration and the possibility to perform weighted fitting for a more reliable analysis. Another good reason to work with AFFINImeter!

 

Why is such a good idea to perform global fitting even for 1:1 interactions?

Advantages of performing a global fitting of several Isothermal Titration Calorimetry isotherms

We have remarked the relevance of global fitting as an indispensable tool to perform a robust analysis of complex binding interactions. Actually, global fitting is a very advantageous tool for the analysis of simpler binding modes like standard 1:1 interactions and it can be used to identify the source of potential differences observed between experiment repeats.
Here we will see how to use global fitting together with the parameters rA and rM of AFFINImeter to identify the source of discrepancies between repeats of the same experiment, to ultimately end up with reliable results.
The isotherms of figure 1 are replicates of the same ITC experiment where a 1:1 interaction between the titrant (A) and the titrate (M) is monitored, but individual fitting of each isotherm to the simple model M + A ↔ MA (Figure 2) yield different KA (association constant) and ΔH values. In fact, from a visual inspection of the isotherms one can presume that  the saturation degree reached in replicate 1 is less than in replicate 2 and that the mid titration point is below the expected stoichiometric equivalence point (At/M= 1), most probably because the titrant (A) and/or titrate (M) concentration was not accurately determined in this experiment.

 

Figure 1. A)  Global fitting 2 where KΔH  and rA are global parameters and rM is individual between isotherms. B) Global fitting 1 where KAΔH and rM are global parameters and rA is individual between isotherms.

 

global fitting of the two isotherms was performed in which KAΔH  and rA are shared fitting parameters amongst the isotherms; moreover, rM* is set as individual fitting parameter to check for potential deviations between nominal and true concentration of M in replicate 1; similarly, a global fitting was performed in which KAΔH and rM are shared parameters, and rA* is an individual fitting parameter to check for potential deviations between nominal and true concentration of (figure 1).

Figure 2. Representation of the experiment using the AFFINImeter AM code.

 

Comparison of the two global fittings clearly shows that a good result is only achieved when deviations in the concentration of A is considered. The value of rA obtained indicates that the active concentration of A is 79% times the nominal concentration (table).

Table: Results obtained from global fitting 2.

 

This protocol using AFFINImeter has already been proven to be useful in drug discovery programs, working with ligand samples of inaccurate concentration.

We would like to thank Dr. Eric Ennifar (University of Strasbourg) for kindly providing the ITC experiments described herein.

 

*rM, rA, are scaling parameters employed to correct for potential differences between the nominal and true concentration of compounds M and A, respectively.

The stoichiometric equilibria approach.

Following the last part of this course, we want to introduce the second fitting approach that can be performed with AFFINImeter:

The Stoichiometric equilibria approach.

This approach uses binding models based on equilibria between stoichiometric species and yields stoichiometric binding constants. In AFFINImeter, traditional and tailored stoichiometric binding models can be easily generated with the “model builder” tool.
Stoichiometric binding models are applicable to a great variety of complex interactions like:

  • A ligand binding to a multi-site receptor with known stoichiometry, where the sites can be independent or cooperative.
  • Competing ligands binding to the same receptor.
  • Oligomerization.

….among others.

The appropriate design and use of binding models in AFFINImeter pass through an understanding of the nomenclature that the software uses to describe the species located in the calorimetric cell and in the syringe.
In the following link you can visualize a quick video about the Nomenclature of the Model model builder:

Nomenclature of Model Builder.

The last video of this course is about how to use the stoichiometric equilibria approach to perform an individual and global analysis.

The course about ITC data analysis comes to an end so if you want to try the versatility of AFFINImeter you can register to get a free trial during 1 month:

 

Get a free trial

Una spin-off de la USC, AFFINImeter, seleccionada por Google para participar en el programa Campus Remote Mentoring.

El Campus Remote Mentoring de Google potencia el crecimiento de nuevas empresas con sesiones de mentorship personalizadas, networking y acceso a nuevos mercados.

AFFINImeter es una spin-off de la Universidad de Santiago de Compostela dedicada al desarrollo de software científico y metodologías para investigación química y farmacéutica en el campo de interacciones moleculares. Entre sus clientes se encuentran diversas farmacéuticas como Bayer, Roche o la filial farmacéutica de Mitsubishi además de prestigiosas universidades de Europa, Japón y Estados Unidos.

El pasado mes de marzo,  AFFINImeter fue seleccionada para participar en el exclusivo programa “Campus Remote Mentoring” de Google, una iniciativa orientada a irradiar la influencia  del campus de Google en Madrid para el resto de start up dentro del territorio español. El programa incluye una tutorización desde la sede de Google en Dublín, y se centra en mejorar aspectos relacionados con el Marketing on-line y la comunicación digital, con el fin de potenciar la internalización de la start up.

Paralelamente AFFINImeter ha sido seleccionada en el prestigioso programa de aceleración de ViaGalicia, promovido por el Consorcio de la Zona Franca y la Xunta de Galicia (a través de la Agencia Gallega de Innovación GAIN y la sociedad de capital-riesgo XesGalicia).

Variable Temperature ITC analysis with AFFINImeter

Analysis of Variable Temperature Isothermal Titration Calorimetry experiments with AFFINImeter

Monitoring a binding event with Isothermal Titration Calorimetry at different temperatures provides a powerful framework for elucidating interesting characteristics of the interaction. Analysis of the isotherms obtained determines the dependence of the association constant (KA) and binding enthalpy (ΔH) with temperature, information that can reveal mechanistic aspects of the interactions, i.e. the existence of allosteric effects and conformational changes (1).

Moreover, kinetic characterization of the interaction at various temperatures gives information about transition state thermodynamics, by means of the dependence of the association and dissociation rate constants (kon and koff) with the temperature. This way, activation free energies of association and dissociation are resolved into its enthalpic and entropic components (2).

Obtaining the full thermodynamic and kinetic profile of 1:1 interactions in a single ITC experiment is now possible with AFFINImeter and KinITC; in order to further exploit the potential of our analytical tools we have recently incorporated a new functionality in AFFINImeter that automatically analyzes variable temperature isothermal Titration Calorimetry assays through Van´t Hoff Plot (Ln(KA) vs 1/T), temperature dependence of ΔH (that determines changes in heat capacity, ΔCp) and Eyring plots (Ln(kon) vs 1/T and Ln(koff) vs 1/T) (3).

AFFINImeter is the only software that provides thermodynamic and kinetic information from a single ITC titration; now incorporates the automatic analysis of variable-temperature experiments.  

You can use this feature for free during one month, go to AFFINImeter webpage.

References

  • Freiburger L, Auclair K, Mittermaier A. Global ITC fitting methods in studies of protein allostery. Methods 2015, 76, pp 149-161.
  • GE Healthcare application note 80. Transition state thermodynamics using Biacore T100, (2007).
  • Ladbury, J. and Doyle, M. (2004). Biocalorimetry 2. Chichester: Wiley.

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!

What you have learned of Isothermal Titration Calorimetry with AFFINImeter in 2015?

AFFINIMETER_NAVIDEÑO_20164cm

What you have learned of Isothermal Titration Calorimetry with AFFINImeter in 2015?

The best gift we can get these holidays is to see that our support has helped your research, leading to improved scientific publications, PhD thesis, and increased research projects successfully accomplished… Happy Holidays!!

AFFINImeter is currently the best  software for the analysis of Isothermal Titration Calorimetry data to obtain a complete thermodynamic and kinetic characterization of molecular interactions. If you are already an user  you are probably aware of all this information, anyway,  we thought that you would like to have a compilation of all the resources we have developed for divulgation and didactic purposes during this year 2015!

For the upcoming year we promise you plenty of new features and improvements in AFFINImeter!

5 Ways in which AFFINImeter can improve your research!

  1. Get more citations from your publications.
  2. Harness the full interpretation of your data
  3. Save time and Material
  4. Get kinetics information from your new and old ITC files
  5. Leave no any ITC isotherm without a fitting. (Read here)

What is the Model Builder?

As you must know with AFFINImeter  complex reaction schemes can be written to describe a real scenario in chemical language. AFFINImeter provides an exclusive “Model Builder” to write the chemical equations describing your system. We have shown you how to take advantage of the model builder.
competitive-interaction-model-ITC

  1. Representative binding models based on a Stoichiometric equilibria and Independent sites approach.
  2. Stoichiometric and site constants: two approaches to analyze data with AFFINImeter.
  3. The stoichiometric equilibria approach to design binding models with AFFINImeter
  4. The independent sites approach to design binding models with AFFINImeter

Guide and tutorial of How to perform a Global analysis with AFFINImeter?

Global fitting reaction parameters 13012015

Complex binding models are usually described by many variable parameters and, in such cases, a single experimental curve is insufficient to achieve a robust analysis. During this year we have prepared several cases of use to show you how to solve easily complex fitting analysis, for instance a global fitting of several ITC isotherms.

KinITC: Get all the kinetic information from your ITC raw data!

We have implemented KinITC, this is a methodology recently developed by Philippe Dumas (CNRS, France) to simultaneously get kinetic and thermodynamic information from a standard ITC experiment. With one single titration experiment it calculates the kinetic constants (kon and koff) and the thermodynamic data (KD and ΔH) of 1:1 binding interactions. The current implementation of kinITC in AFFINImeter is valid only for 1:1 interactions but we intend to extend this for more complex systems in the near future.

Did you miss our Webinars? Here you can get the slides of the presentations!

Malvern Webinar:

AFFINImeter was guest presenter of the Malvern webinar on advanced Isothermal Titration Calorimetry (ITC) data analysis. (Read more).

 UNAM webinar:
The following slides corresponds to different webinars that we have done for research groups interested in learning more about AFFINImeter.

Selected AFFINImeter publications

Tecnologías de vanguardia en biomedicina: Orbitrap (ion trap mass analyzer) y SPR (Surface Plasmon Resonance

La Dr. Eva Muñoz, miembro del staff científico de S4SD ha sido invitada para participar en el seminario  “Tecnologías de vanguardia en biomedicina: Orbitrap (ion trap mass analyzer) y SPR (Surface Plasmon Resonance)” que tendrá lugar el día 16 de Diciembre en Vigo. El seminario es organizado por el Instituto de Investigación Biomédica de Ourense-Pontevedra-Vigo, en el marco del proyecto BIOCAPS

Al seminario también  están invitados la Dr. Mónica Carrera (Instituto de Investigaciones Marinas de Vigo, IIM-CSIC) experta en proteómica, el Dr. Manuel Marcos (Universidad de Vigo, CACTI) para hablar de aplicaciones de  Orbitrap (Ion trap mass analyzer) y la Dra. Pilar Canoa (Universidad de Vigo, CINBIO) para hablar de aplicaciones de SPR (Surface plasmon resonance).

La inscripción es gratuita y está abierta hasta el día 14 de Diciembre de 2015.
Cartel-seminario-BIOCAPS

Don’t miss the AFFINImeter black Friday deal!

 


Don’t miss the AFFINImeter black Friday deal!

This Black Friday our Scientific Staff puts their expertise at your service to perform a complete analysis of you ITC data.

From 27-11-2015 to 30-11-2015

AFFINImeter Black Friday

 

 

  • Send us an email of interest  to support@affinimeter.com with the subject “AFFINImeter black Friday deal” and our Scientific staff will contact you to learn about your particular problem.
  • We will use all the potential of AFFINImeter to get kinetic and thermodynamic information of your interaction: KinITC, advanced binding models and global fitting.
Request your deal

Be fast!

Our deal is limitted to the first 20 requests.

We will send you a detailed report with the results within the following days.

 

We will be attending the European MicroCal Users’ Meeting 2015 organized by Malvern Instruments

On November 24th and 25th will be attending the  European MicroCal Users’ Meeting 2015 organized by Malvern Instruments. 

Our sales representant Dr. Juan Sabín will be there sharing ideas on new applications and challenges on Isothermal Titration Calorimetry (ITC) experiments and data analysis. We will be pleased to talkt to you. Contact us.

We are sharing the Workshop agenda publised  in the Malvern website where you will find all information of the Workshop and registration.

Free Morning Workshop
8:30 Registration and coffee
9:00 “Recent developments in interaction analysis: introducing the new MicroCal PEAQ-ITC”
Dr. Marco Marenchino, Applications Specialist, Malvern Instruments
10:30 Coffee break
11:00 “Keep your DSC at top performance: experiment design and advanced data analysis”
Dr. Marco Marenchino, Applications Specialist, Malvern Instruments
12:00 Lunch
Meeting Opens 
13:00 “Opening remarks”
Dr. Mark Wingfield, General Manager GmbH, Malvern Instruments
Plenary Lecture
13:10 “Profiling Enthalpy/Entropy to Aid Decision-Making in Drug Optimization: Fact or Fantasy?”
Prof. Gerhard Klebe, University of Marburg
Calorimetry for the studies of protein-ligand interactions in academia and DD industry
13:50 Microcalorimetry with small molecule compounds at Merck-Serono
Dr. Ansgar Wegener, Merck
14:20 “Affinity and mechanostability in protein-protein interactions”
Lukas Milles M.Sc. and Magnus Bauer M.Sc. LMU
14:50 Coffee break
15:10 “Thermodynamic landscape of the bacterial 30S translation initiation complex assembly”
Dr. Eric Ennifar, Research Director at CNRS, University of Strasbourg
15:40 The metal-dependent network for nickel delivery into urease: protein interactions and conformational changes
Dr Barbara Zambelli, University of Bologna
16:10 Drug-target interactions put under the microscope
Mrs Barbara Wienen, University of Marburg
16:40 ‘How to be PIQy’
Dr. Chris Johnson, MRC Laboratory of Molecular Biology, Cambridge
19:00 Calorimetric Dinner

November 25th

Protein and antibody characterization
08:30 “Current Applications of ITC in antibody design and development”
Christian Gassner, Roche Diagnostics
09:00 “Lessons learned from pH and excipient screening for mAb formulation development by using DSC”
Dr. Tim Menzen, Coriolis Pharma
09:30 “Accelerating biotherapeutic development”
Dr. Marco Marenchino, Application Specialist, Malvern Instruments
10:00 “DSC in formulation development of insulins”
Dr. Ursula Stock, Sanofi-Aventis
10:30 Coffee break
Data Analysis
11:00 “Complex binding models and Global Fit”
Dr. Natalia Markova, Principal Scientist – MicroCal, Malvern Instruments
11:30 “High precision ITC with automated peak-shape analysis”
Prof. Dr. Sandro Keller, Molecular Biophysics, University of Kaiserslautern,Germany
12:00 Lunch
Novel Applications I
13:00 “Microcalorimetric studies of lipid and detergent systems”
Prof. Dr. Heiko Heerklotz, Professor for Pharmaceutics, University of Freiburg
13:30 “Enzymatic conversion of biomass: A calorimetric approach”
Prof. Dr. Peter Westh, Professor of Biophysical Chemistry, Roskilde University
14:00 “Nanotools to understand when a protein can induce an immune response”
Dr. Mihaela Delcea, Group Leader Nanostructure, University of Greifswald
14:30 Coffee break
Novel Application II
15:20 Calorimetric studies of supramolecular peptide assembly and protein aggregation
Prof Büll, University of Düsseldorf
15:50 “KinITC: Get more out of ITC data”
Dr. Eric Ennifar, Research Director at CNRS, University of Strasbourg
16:20 “Concluding remarks”
Dr. Mark Wingfield, General Manager GmbH, Malvern Instruments