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

Why Microcalorimetry? Heat is generated or absorbed in every chemical processIn-solutionReal-Time & Direct measurements No molecular weight limitationsLabel-freeNo optical limitations

Calorimetry in the Life SciencesBinding Studies Quick and accurate affinitiesMechanism of action and conformational changesStructure-function relationshipsSpecific vs. non-specific binding Kinetics KM, Vmax, kcat Enzyme Inhibition

How Do They Work?Measuring Temperature Changes in CalorimetryReference 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: AffinitiesBinding mechanism Number of binding sitesKinetics

Range of Binding: KA = 102 1010 M-1

Isothermal Titration CalorimetryTypical ITC Data

Range of macromolecule concentration in the cellC-value (unitless constant) Ka binding (association) constantM tot the total macromolecule concentration in the celln stoichiometry parameterc-value = Ka Mtot n Working Range: c-value 5 to 500Ligand conc. = 10 n Mtot

Enthalpic and Entropic Contributions to Binding Affinity Enthalpy and Entropy make up the affinity (G=-RTlnKa)

G = H - TS

Binding MechanismSame affinity but different binding mechanisms and specificity

Enthalpy and Entropy EntropyHydrophobic interactionsWater release Ion release Conformational changes EnthalpyHydrogen bonding Protonation eventsMore specific

Energetic SignaturesA is enthalpy driven. Strong H-bondingV.d. Waals interactions coupled to a conformational change

B is entropically driven Hydrophobic Interactions and possibly rigid body

C is mildly enthalpic and entropicSmall 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 ProteaseOhtaka, et al. Protein Sci. 11, 1908-1916 (2002)

ITC Protein-Protein InteractionA: Wild-type cytochrome c titrated into wild-type cytochrome c peroxidase

B: Mutant cytochrome c titrated into mutant cytochrome c peroxidasePielak and Wang, Biochemistry 40, 422-428 (2001)

Schematic representation of the regulatory and the induction mechanism of TtgRTtgR blocks the access of the RNA-POL by binding to the overlapping ttgR-ttgABC operator regionBinding of a ligand to TtgR induces a conformational change and releases it from the DNARNA-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

NaringeninQuercetinPhloretinAt least one aromatic ring is the common feature of the ligands

TtgR in complex with Naringenin and Chloramphenicol3.2 l aliquots of 1mM naringenin into native 50M monomer TtgR (I) and into 50M monomer mutant TtgRV66A/L96A

Effector

Protein

KA

(M -1)

KD

(M)

G

(kcal/mol)

H

(kcal/mol)

TS

(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 0.1

TtgRL66A/V96A

(9.11.1) 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 R176Buffer:25mM PIPES, 250mM NaCl, 5% Glycerol, 10mM MgAc, 10mM KCl

The role of R176 in ligand recognition of TtgRP. putida DOT-T1E AAVAMFAYVDGLIRRWLL 180P. putida KT2440 AAVAMFAYVDGLIGRWLL 180

Two binding sites exhibit positive cooperativity

Effector

Protein

KA

(M -1 )

KD

(M)

G

(kcal/mol)

H

(kcal/mol)

TS

(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

Titration of three DNA double-strand oligomers of the wild-type operator with TtgRInjection of 6 l aliquots of 40-wt (18.8 M) into 8.1 M TtgR (dimer) Injection of 8 l aliquots of 30-wt (26.5 M) into 10.2 M TtgRInjection of 16-l aliquots of 14 M 28-wt into 6.1 M TtgR40bp-wt: kD = 1.57( 0.04) MH = 6.33( 0.03) kcal/mol

30bp-wt: kD = 1.23( 0.05) M H = 5.95( 0.04) kcal/mol

28bp-wt: no binding

Practical considerationsTypical (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 SummaryAffinities and Binding Energetic profile of reactionMechanisms of Binding StoichiometryEnzyme Kinetics