ITC eksamensnotater

Almost any chemical reaction or physical change is accompanied by a change in heat,

or enthalpy, whether the heat is taken up from the surroundings (endothermic reaction)
or released to the surroundings (exothermic reaction)

Calorimetric measurements can be made in 3 different ways: temperature change,

power compensation (isothermal) or heat conduction

Isothermal titration calorimetry is a technique allowing the direct measurement of the

magnitude of the binding affinity, and the magnitude of the two thermodynamic terms
that define the binding affinity: the changes in enthalpy H, and entropy S.

H, enthalpy is indication of changes in hydrogen and van der Waals bonding

-TS, entropy is indication of changed in hydrophobic interactions and

conformational changes.

N, stoichiometry indicated the ratio of ligand to macromolecule binding (number of

binding seats)

The experiment:
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A reaction cell is filled with one of the partners, B

An injection syringe contains the other compound, A, which will be released in the
reaction cell by means of small injections, allowing time for the reaction to happen..

The binding reaction releases or absorbs heat from the environment.

Since the system is maintained at a constat temperature. The instrument registers the
heat difference between the reference cell and the reaction cell, and compensates by
reducing or increasing the power applied to the control heater, so that the temperature
remains constant

The instrumental response is the amount of power (microcal or microjoules per

second) necessary to maintain constant temperature between the reaction and
reference cells.

The raw signal in the power compensation calorimeter is the power J/sec applied to
the control heater that is required to keep the calorimeter cell from changing
temperature as a function of time. The heat change is then simply calculated by
integrating the heater power over the time of the measurement

The more injections that are done with ligand from the syringe into the protein
solution in the reaction cell, the more saturated the protein becomes. Less P is
available for binding, less complex is formed and the heat of the reaction decreases.

When al P is found in complex with ligand, no more heat is generated.

The data received from the experiment are fitted by non- linear regression

An ITC experiment give access to parameters such as the free energy of the bidning,
the association and dissociation constants, the enthalpy and entropy of the reaction.

Thermodynamic parameters, but not kinetic (rate constants or rates)

Blank data of dilution effects and mechanical effects of injecting ligand into buffer
must be made to determine the background level of heat change.

Planning ITC experiment

Planning experiment, doing simulations

Preparing solutions

Collection raw ITC data

Collecting blank data

Correcting raw ITC data

Nonlinear regression of the corrected titration data to provide estimates of the

thermodynamic parameter values

Interpretation of model data

Buffers:
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Use salt concentration an pH where the compounds are stable and soluble.

Degas all buffers and solutions

The enthalpy associated to the protonation, heat of ionization, vary according to the
buffers

Use buffers that have a heat of ionization that is close to zero, as small as possible.

This includes phosphate, acetate an sulphate buffers, but not Tris buffers

DTT should be avoided as it is unstable

Avoid tris buffers when working at different temperatures

Does the system adopt different conformations at different pH?

Advantages

NO labels

In solution

No molecular weight limitations

Easy procedure

Drawbacks:
-

ITC requires a large amount of material, protein and ligand.

Preparation
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All buffers and solutions must be degassed to avoid bubbles or air that can interfere
with the heat change (create heat)

The major advantage of ITC derives from the fact that the binding isotherms are
defines in terms of the heats of reaction and as such they allow a direct estimation of
enthalpy changes in addition to the association constant.

Thus a single calorimetric titration provides a complete characterization of the

energetics of binding.

C- values: C = [M] * Ka, concentration of macromolecule..? and Kd is the association

constant of the reaction. C should be between 10-50 to obtain good curves.

Ligand concentration should be around 10-20 * [M] protein

Theory
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More negative G, higher affinity

H measure of the energy content of the bonds broken and created. The dominant
contribution is from hydrogen bonds. Negative value indicates enthalpy change
favoring the binding

Solvents play a role

S, entropy, is positive for entropically driven reactions. The major contributions are
from hydrophobic effects and solvation effects, such as release of the ordered water
shell upon binding.

Any non-specific effects (buffer mismatch, pH mismatch, heat of dilution, heat of

ligand dissociation)
All the following situations can apply for binding that has the same free energy and
affinity:

A. Good hydrogen bonding with unfavorable conformational change

B. Binding dominated by hydrophobic interaction C. Favorable hydrogen bonds and

hydrophobic interaction

ITC exam questions:

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What events are responsible for the heat release or absorption in a protein-ligand
binding

What is the magnitude of these forces?

Draw a typical figure of the raw data generated during an exothermic protein-ligand
reaction

How do you choose a suitable buffer to study protein- ligand interactions with ITC?