Wei Li

Department of Chemistry

University of Victoria

Winter, 2013

Measurement of Binding Constants and Heats of Binding using Isothermal Titration Calorimeter

Acknowledgement

Professor Hof

Classmates

Outline

• Thermodynamic parameters• Isothermal titration calorimeter (ITC)• ITC development history• Advantages of using ITC• Basic configuration of an ITC• Sample preparation• Raw data and plot of processed data• Systematic errors• References

Thermodynamic Parameters

When characterizing interactions between a biological macromolecule M and a small ligand X, or between two macromolecules,

M + X = MX

MX + X = MX2

…….

MXn-1 + X = MXn,

K = [MXn]/[MXn-1] X]

G = G° + RTlnK (Standard conditions: 1 mole of P and 1 mole of L at PH7 and 25° C)

At equilibrium, under standard conditions, G = 0.

G° = -RTlnK = H° - TS°

The single-site binding constant K, H°,and the number of sites n are the independent variables of thermodynamicinterest.

G° and S° of binding are dependentvariables obtained by the calculation.

Thermodynamic Parameters

Entropy• Hydrophobic

interactions• Water release • Ion release • Conformational changes

Enthalpy• Hydrogen bonding • Protonation events

n = stoichiometry

Number of protein binding sites reflects the purity and the functional integrity of a protein preparation if the measure and fitted stoichiometry can be compared to the known, previously determined stoichiometry

ITC Development History

• 1960s – built in the second half to study chemical reactions• 1970s – sensitivity: mJ

metal + ligand complex• 1980s – sensitivity: µJ

ligand binding processes and micelle formation

• 1990s – number of published papers increased due to new commercial calorimeters became available. Evolved from a specialist method to a widely used technique

• 2000s – widely employed in the design and discovery of new drugs and for the study of liquid mixtures

• 2009 – 374 out of 432 papers on protein interaction with other proteins, small molecules, metal ions, lipids, nucleic acids, and carbohydrates as well as on nucleic acid interactions with small molecules.

Advantages of using ITC

No labeling required In-solution No molecular weight limitations Optical clarity unimportant Minimal assay development Using heat as signal In one single experiment, K, ∆H, and Stoichiometry n of

interaction between two or more molecules in solution can be determined

∆G and ∆S can be calculated

Mark A. Williams and Tina Daviter (eds), Protein-Ligand Interactions: Methods and Applications. Springer Science and Business Media, New York, 2013

Basic Configuration of an ITC

• Reference cell and sample cell in an adiabatic jacket. • Reference cell filled with buffer or water. Sample cell filled with protein.• Both cells are heated in such a way that the temperature is almost

constant, i.e. T < 10 x (E-6) ° C at all times.• Syringe device holds the ligand. This device titrates the ligand into the

sample cell and also acts as a stirrer.

“Isothermal titration calorimetry (ITC) is a biophysical technique that allows a thermodynamic characterization of an interactive system”.

VP-ITC micro calorimeter

Basic Configuration of an ITC

• Reference and sample cells containing identical buffer are located within an adiabatic jacket, and the latter contains macromolecule of interest.

• A small, constant power is applied to the reference cell, which activated the cell feedback circuit to drive ∆T to 0.

• No reaction – the feedback power remains constant at the resting baseline value.

• Exothermal reactions temporarily ↓ and endothermic reactions temporarily ↑ feedback power. The reaction heats are readily obtained by computer integration of these deflections from the resting baseline.

Sample Preparation

General consideration• Experiment performed at reasonable PH• Protein has to be extensively dialysed and the ligand has to be dissolved

in the buffer recovered from the last protein dialysis step

Protein preparation• As pure as possible• Concentration should be correctly estimated.

Ligand preparation• As pure as possible• Accurate concentration

Buffers

Buffers with low ionization enthalpy should be considered.

Raw Data and Plot of Processed Data

• Top: Raw ITC data showing an exothermic binding reaction. Each peak corresponds to an injection of ligand into a protein solution in the sample cell; the area under the peak is proportional to the amount of heat released in the binding reaction.

• When the protein becomes saturated, the DP signal diminishes until only the background heat of dilution is observed.

• Bottom: Binding isotherm obtained by integrating the area of each peak. The heat released per mole of ligant is plotted against the molar ratio of the two reactants.

Systematic Errors

Blank experimentHeat of dilution of ligandHeat of dilution of macromoleculeBuffer to buffer experimentHeat of proton ionization

Degassing

Generation of bubbles during an ITC experiment will generate spurious heat signals.

Measurements of the concentrations

Accuracy of the measurements are imperative for a good ITC experiment.

Sample concentrations also need to be within a proper range to get reliable data.

Small molecules impurities and PH mismatches in the buffer will cause artifacts.

References

• Nunez, S.; Venhorst, J.; Kruse, C. G. Target–drug interactions: First principles and their application to drug discovery. Drug Discov. Today 2012, 17, 10–22.

• Wiseman T. et al, Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179: 131-137, 1989.

• Mark A. Williams and Tina Daviter (eds), Protein-Ligand Interactions: methods and applications. Springer Science and Business Media, New York, 2013.

• Joel Tellinghuisen, John D. Chodera, Systematic errors in isothermal titration calorimetry: concentrations and baselines, Analytical Biochemistry 414 (2011) 297-299.

• wikepedia

Transferrin Human serum albumin