Protein interaction studies using Isothermal titration calorimetry (ITC) Yilmaz Alguel.

Protein interaction studies using Isothermal titration

calorimetry (ITC)

Yilmaz Alguel

Why Microcalorimetry?

• Heat is generated or absorbed in every chemical process

• In-solution• Real-Time & Direct measurements • No molecular weight limitations• Label-free• No optical limitations

Calorimetry in the Life Sciences

Binding Studies • Quick and accurate affinities

• Mechanism of action and conformational changes

• Structure-function relationships

• Specific vs. non-specific binding

Kinetics • KM, Vmax, kcat Enzyme Inhibition

How Do They Work?

• Measuring Temperature Changes in Calorimetry

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Reference cell contains H2O(can be filled with Buffer as well)



ITC: A Method for Characterizing Binding Interactions

• Mixture of two components at a set temperature

• Heat of interaction is measured

Parameters measured from a single ITC experiment:

• Affinities• Binding mechanism • Number of binding sites• Kinetics

Range of Binding: KA = 102 – 1010 M-1

Isothermal Titration Calorimetry

• Typical ITC Data



Range of macromolecule concentration in the cell

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C-value (unitless constant) Ka binding (association) constantM tot the total macromolecule concentration in the celln stoichiometry parameter

c-value = Ka Mtot n

Working Range: c-value 5 to 500

Ligand conc. = 10 n Mtot

Enthalpic and Entropic Contributions to Binding Affinity

• Enthalpy and Entropy make up the affinity (G=-RTlnKa)

G = H - TSQuickTime™ and a

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Binding Mechanism

• Same affinity but different binding mechanisms and specificity



Enthalpy and Entropy

• Entropy• Hydrophobic interactions

• Water release

• Ion release

• Conformational changes

• Enthalpy• Hydrogen bonding

• Protonation events

• More specific



Energetic Signatures

A is enthalpy driven. • Strong H-bonding• V.d. Waals interactions • coupled to a conformational change

B is entropically driven • Hydrophobic • Interactions and possibly ‘rigid body’

C is mildly enthalpic and entropic• Small negative or positive enthalpy • (expulsion of structured H2O molecules from the binding site)• (Releasing H2O would increase entropy)



Drug Discovery –Binding of Inhibitors to HIV-1 Protease



Ohtaka, et al. Protein Sci. 11, 1908-1916 (2002)

ITC –Protein-Protein Interaction

• A: Wild-type cytochrome c titrated into wild-type cytochrome c peroxidase

• B: Mutant cytochrome c titrated into mutant cytochrome c peroxidaseQuickTime™ and a

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Pielak and Wang, Biochemistry 40, 422-428 (2001)

Schematic representation of the regulatory and the induction mechanism of TtgR

TtgR blocks the access of the RNA-POL by binding to the overlapping ttgR-ttgABC operator region

Binding of a ligand to TtgR induces a

conformational change and releases it from the DNA

RNA-POL is able to bind the ttgR-ttgABC operator and transcribe the efflux pump genes ttgABC and the

ttgR repressor gene encoded divergently

TtgR-binding antibiotics and plant antimicrobials

Chloramphenicol Tetracycline

NaringeninQuercetin Phloretin

At least one aromatic ring is the common feature of

the ligands

TtgR in complex with Naringenin and Chloramphenicol





3.2 l aliquots of 1mM naringenin into native 50M monomer TtgR (I) and into 50M monomer mutant TtgRV66A/L96A

Effector Protein KA

(M -1)

KD

(µM)

∆G

(kcal/mol)

∆H

(kcal/mol)

T∆S

(kcal/mol)

Naringenin TtgR (5.5 ± 0.1) x 104 18 ± 0.3 -6.6 ± 0.1 -10.8 ± 0.1 -4.2 ± 0.1

TtgRL66A/V96A (2.0 ±0.1) x 104 49 ± 2 -6.0 ±0.2 -5.3 ± 0.2 0.7 ± 0.2

Chloramphenicol TtgR (3.1 ±0.3) x 104 32 ± 3 -6.2 ± 0.9 -7.0 ± 1.0 -0.8 ± 0.1TtgRL66A/V96A (9.1±1.1) x 103 109 ± 13 -5.5 ±1.5 -3.5 ± 0.9 2.0 ± 0.9

Titration of TtgR with Phloretin & the role of residue R176

Buffer:25mM PIPES, 250mM NaCl, 5% Glycerol, 10mM MgAc, 10mM KCl

The role of R176 in ligand recognition of TtgR



Titration of TtgRR176G with phloretin

P. putida DOT-T1E …AAVAMFAYVDGLIRRWLL… 180P. putida KT2440 …AAVAMFAYVDGLIGRWLL… 180

Crystal structure of TtgR and mutant TtgRR176G with phloretin

Two binding sites exhibit positive cooperativity

Effector Protein KA

(M -1 )

KD

(µM)

∆G

(kcal/mol)

∆H

(kcal/mol)

T∆S

(kcal/mol)

Phloretin TtgRa (2.1 ±0.4) x 107

(4.6 ±1.1) x 105

0.05 ± 0.01

2.2 ± 0.5

-10.2 ± 0.1

-7.8 ± 0.2

-15.1 ± 0.1

-1.8 ± 0.2

-4.9 ± 0.2

6.0 ± 0.2

TtgRR176G (1.09 ± 0.06) x 105 9.2 ± 0.5 -7.0 ± 0.1 -21.6 ± 0.5 -14.6 ± 0.2

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Titration of three DNA double-strand oligomers of the wild-type operator with TtgR

(A) Injection of 6 μl aliquots of 40-wt (18.8 μM) into 8.1 μM TtgR (dimer) (B) Injection of 8 μl aliquots of 30-wt (26.5 μM) into 10.2 μM TtgR(C) Injection of 16-μl aliquots of 14 μM 28-wt into 6.1 μM TtgR

40bp-wt: kD = 1.57( ± 0.04) μMΔH = 6.33( ± 0.03) kcal/mol

30bp-wt: kD = 1.23( ± 0.05) μM ΔH = 5.95( ± 0.04) kcal/mol

28bp-wt: no binding

Practical considerations• Typical (macromolecule) concentrations down to 10 micromolar(e.g. 0.25 mg/ml for

a 25kDa protein) in the reaction cell (1.5ml volume), with 15-20x higher concentrations of titrant(ligand) in the injection syringe (min. 300 microlitrerequired).

• At the end of the titration, typically 250ul of ligand will have been added to 1.5ml of macromolecule.

• Both macromolecule and ligand must be in identical buffer/solvent otherwise large heats of dilution will mask the desired observation.

• Dialysis of the macromolecule against appropriate buffer, using the final dialysis buffer to make up the ligand solution.

• Truly quantitative data can only be obtained if molarcon centrations of proteins/macromolecules and ligands are known accurately. This can usually be done UV/visabsorbance measurements, provided molar extinction coefficients are available.

Microcalorimetry Summary

• Affinities and Binding

• Energetic profile of reaction

• Mechanisms of Binding Stoichiometry

• Enzyme Kinetics

Protein interaction studies using Isothermal titration calorimetry (ITC) Yilmaz Alguel.

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