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

2. 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

3. 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

4. How Do They Work? Measuring Temperature Changes in Calorimetry Reference cell contains H 2 O (can be filled with Buffer as well)

6. 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: K A = 10 2 – 10 10 M -1

7. Isothermal Titration Calorimetry Typical ITC Data

8. Range of macromolecule concentration in the cell C-value (unitless constant) Ka binding (association) constant M tot the total macromolecule concentration in the cell n stoichiometry parameter c-value = K a M tot n Working Range: c-value 5 to 500 Ligand conc. = 10 n M tot

9. Enthalpic and Entropic Contributions to Binding Affinity Enthalpy and Entropy make up the affinity (  G=-RTlnKa)  G =  H - T  S

10. Binding Mechanism Same affinity but different binding mechanisms and specificity

11. Enthalpy and Entropy Entropy Hydrophobic interactions Water release Ion release Conformational changes Enthalpy Hydrogen bonding Protonation events More specific

12. 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 H 2 O molecules from the binding site) (Releasing H 2 O would increase entropy)

13. Drug Discovery –Binding of Inhibitors to HIV-1 Protease Ohtaka, et al. Protein Sci. 11, 1908-1916 (2002)

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

15. 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

16. TtgR-binding antibiotics and plant antimicrobials Chloramphenicol Tetracycline Naringenin QuercetinPhloretin At least one aromatic ring is the common feature of the ligands

17. TtgR in complex with Naringenin and Chloramphenicol 3.2  l aliquots of 1mM naringenin into native 50  M monomer TtgR (I) and into 50  M monomer mutant TtgRV66A/L96A

18. Titration of TtgR with Phloretin & the role of residue R176 Buffer:25mM PIPES, 250mM NaCl, 5% Glycerol, 10mM MgAc, 10mM KCl

19. The role of R176 in ligand recognition of TtgR Titration of TtgR R176G with phloretin P. putida DOT-T1E …AAVAMFAYVDGLIRRWLL… 180 P. putida KT2440 …AAVAMFAYVDGLIGRWLL… 180 Crystal structure of TtgR and mutant TtgR R176G with phloretin

20. Two binding sites exhibit positive cooperativity

21. 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) μM ΔH = 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

22. 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.

23. Microcalorimetry Summary Affinities and Binding Energetic profile of reaction Mechanisms of Binding Stoichiometry Enzyme Kinetics