Isothermal Titration Calorimetry Flashcards
What are the two main uses of Isothermal Calorimetry in the life sciences?
Binding studies
Kinetic studies
What can we learn about binding?
- Quick and accurate affinities
- Mechanism of action and conformational changes
- Structure-function relationships
- Specific vs. non-specific binding
What is isothermal titration calorimetry ITC?
The protein in the measuring cell is kept at a constant temperature.
The ligand is added stepwise from a syringe and the heat which is released or taken up due to binding is compensated for by a sensitive thermostat.
The electrical power of the thermostat is directly related to the enthalpy of the reaction.
Exothermic ligand binding causes a _ peak in the signal
Negative
A standard ITC trace has two panels, what does the top panel show?
The heat trace of the thermostat over the time of the experiment with the individual injections of ligand as peaks.
A standard ITC trace has two panels, how is the data in the lower panel obtained?
By integrating the area of the peaks and plotting them against the molar ratio of ligand and protein.
How to obtain the binding isotherm?
By fitting a quadratic binding curve to the data
How are affinity and the dissociation constant related
Inversely
What is microcalorimetry?
A method of studying biological interactions in solutions using relatively low amounts of protein and ligand.
What can microcalorimetry tell us?
The precise determination of stoichiometry of binding reactions
Gives direct access to all thermodynamic parameters from one single experiment
Which ligands can be studied using microcalorimetry?
It is applicable to ligands such as proteins, peptides, lipids, liposomes, DNA, ions,…
What is the main requirement for ITC?
Purified protein
(we also need the exact molecular weight)
Why are dimerization interactions problematic?
Because it is a binding reaction and so will have an accompanying enthalpic change
How might you use ITC for drug discovery?
Test a library of compounds (eg. Inhibitors) to a protein to determine the one with the greatest specificity.
What factors impact the kinds of isotherms?
- Exothermic or Endothermic=
- Influence by buffer conditions
- Nature of the ligand or protein binding site.
How do you work out how many binding sites there are?
If you think you know how many binding sites you have, the computer can fit your data to the corresponding curve. Want to optimise Chi Squared value.
Data obtained from ITC
- Number of binding sites.
- Affinity for each site ΔG = -RTlnKa
- ΔH – the overall change in enthalpy for the binding event
- -TΔS – the overall change in entropy for the binding event
What factors effect entropy in ITC?
- Hydrophobic interactions
- Water release
- Ion release
- Conformational changes
What factors affect enthalpy in ITC?
- Hydrogen bonding
- Protonation events
- More specific
Entropy driven binding
Usually associated with exothermic binding where there is considerable disorder in the water molecules as a result of the binding to the protein.
Usually associated with hydrophobic interactions with the protein.
In drug design ITC plays a role in the testing of _ of ligand libraries to a target.
binding
What do high entropic isotherms suggest?
non-specific interactions, hydrophobic interactions but also suggest no loss of conformational freedom
What is a good result from a binding study of ligands in drug design?
a contribution of both enthalpic and entropic components to G – this is optimal
Protein binding metals
Copper, manganese, iron, nickel, zinc
