1. 10 good reasons to perform ITC experiments
Structure & Dynamics of Molecular Machines
Eric ENNIFAR
CNRS
Strasbourg

2. What is ITC ? ITC = Isothermal Titration Calorimetry
Most intermolecular interactions are followed by heat generation or absorption.
ITC is a thermodynamic technique that directly measures the heat released or absorbed upon a binding event, thus allowing an accurate determination of:
- binding constant (Ka)
- reaction stoichiometry (n)
- enthalpy (DH)
- entropy (DS)
Complete thermodynamic profile of the molecular interaction
in one single experiment

3. What is ITC ?
Endothermic (DH>0)
Exothermic (DH<0)

4. What is ITC ? Benefits from ITC:
- true in-solution technique: no labelling/immobilization of the sample is required
- no lower or upper molecular weight limitations (applicable from simple diatomic complexes to MDa complexes)
- no buffer/salt restriction
- direct measurement of sub-millimolar to nanomolar binding constants
- sample can be recovered
Drawbacks:
- requires large amount of sample
Applications:
Characterization of molecular interactions
Ligand screening
Enzyme Kinetics
Evaluation of the biological activity
More…

5. How does ITC work ?
An optimal determination of the Kd is obtained if 500 > c > 10
where c = number of sites x Ka x sample concentration
An optimal determination of the DH is obtained if the initial plateau is observed during the experiment
c=1000
c=100
c=1

6. How does ITC work ? DG = DH - TDS DCp = (DH/T)
DS (entropy) and DG (free Gibbs energy) are obtained from the observed Kd (dissociation constant) and DH (enthalpy)
DG = - RT ln (1/Kd)
DG = DH - TDS
DCp = (DH/T)

7. HIV-1 reverse transcriptase
Quality Control
HIV-1 reverse transcriptase +
Primer/template DNA + A
DLS B
Batch with folding problem
SDS PAGE MS
DH = ± 0.15 kcal/mol
DS = 104 cal/mol/deg
N = 0.64
Kd = 46 ± 7 nM
DH = ± 0.17 kcal/mol
DS = 104 cal/mol/deg
N = 0.95
Kd = 53 ± 5 nM

8. Monitor step by step successive chemical steps in molecular machines
Incremental-ITC
Monitor step by step successive chemical steps in molecular machines

9. HIV-1 reverse transcriptase
Monitoring of biological activity
HIV-1 reverse transcriptase +
Primer/template DNA +
Primer/template 1
Primer/template 2
100% active
Thermodynamic signature of the active RT/DNA complex
Kd = 59 ± 9 nM
DH = 23.4 ± 1.4 kcal/mol
DS = 116 cal/mol/K
Catalytically-incompetent binding mode

10. Monitoring of biological activity
NVP

11. Kinetic ITC: kinITC
Kon, koff

12. vs 5'-AAG AAG AGG AG-3' 3'-TTC TTC TCC TC-5' NikR-operator / DNA
Kinetic ITC: kinITC vs
5'-AAG AAG AGG AG-3'
3'-TTC TTC TCC TC-5'
kon M-1s M-1s-1
koff s s-1
NikR-operator / DNA
kon M-1s M-1s-1
koff s s-1

13. Bacterial 70S ribosomal initiation complex formation
MW limitations: no limits !
Bacterial 70S ribosomal initiation complex formation
30S-IF2/GTP-mRNA-tRNAfMet,i +
50S }
50S
monodisperse
QC using DLS
30S

14. Bacterial 70S ribosomal initiation complex formation
MW limitations: no limits !
Bacterial 70S ribosomal initiation complex formation
30S-IF2/GTP-mRNA-tRNAfMet,i +
50S
N = 0,41
∆H = -178,7 ± 3,1 kcal/mol
∆S = -570 cal/mol/deg
Kd = 361 ± 31 nM

15. MW limitations: no limits !
1.7 kDa
Bac7(1-16)
antibiotic
N= 1.22
Kd = 217 nM
∆ H = kcal/mol
-T∆ S = 7.1 kcal/mol
∆ G = -9.4 kcal/mol
2.4 MDa
70S Ribosome

16. MW limitations: no limits !
96 kDa
CrPV IRES RNA
N= 1.08
Kd = 48 nM
∆ H = kcal/mol
∆ S = cal/mol/deg
3.3 MDa
Yeast 80S Ribosome

17. Truly label-free ! antibiotics Erythromycin Bac7(1-16) Azithromycin
Kd = 217 nM
∆ H = kcal/mol
-T∆ S = 7.1 kcal/mol
∆ G = -9.4 kcal/mol
N= 1.17
Kd = 40 nM
∆ H = -1.3 kcal/mol
-T∆ S = -8.5 kcal/mol
∆ G = -9.8 kcal/mol
N= 0.73
Kd = 1 nM
∆ H = -3.0 kcal/mol
-T∆ S = -9.0 kcal/mol
∆ G = kcal/mol
70S Ribosome

18. Analysis of molecular forces involved in binding
antibiotics
Erythromycin
Bac7(1-16)
Azithromycin
N= 1.22
Kd = 217 nM
∆ H = kcal/mol
-T∆ S = 7.1 kcal/mol
∆ G = -9.4 kcal/mol
N= 1.17
Kd = 40 nM
∆ H = -1.3 kcal/mol
-T∆ S = -8.5 kcal/mol
∆ G = -9.8 kcal/mol
N= 0.73
Kd = 1 nM
∆ H = -3.0 kcal/mol
-T∆ S = -9.0 kcal/mol
∆ G = kcal/mol
70S Ribosome
∆ H
∆ S
∆ G

19. HIV-1 reverse transcriptase
Heat-capacity change DCp
HIV-1 reverse transcriptase +
Primer/template DNA +

20. HIV-1 reverse transcriptase
Heat-capacity change DCp
HIV-1 reverse transcriptase +
Primer/template DNA +
-1.14 < DCp < -0.74
Negative DCp: local folding coupled to binding
DH = 34°C (van’t Hoff) extremum of the Kd when DH = 0

21. Riboswitch/ligand interaction
ITC-guided crystallization
Riboswitch/ligand interaction
Kd = 33 nM
DH = kcal/mol
DS = cal/mol/K
N = 0.58
Problem !
Clear Drops …
N = 1.01
Native acrylamide gel revealed 2 conformations of the RNA !
New RNA folding protocol

22. + ITC-guided sample preparation for cryoEM
IF3
30S ribosome
30S-IF3 8 Å resolution

23. Architecture et Réactivité de l’ARN
Reverse Transcriptase
Architecture et Réactivité de l’ARN
Structure & Dynamics of Molecular Machines
Benoit MEYER
Guillaume BEC
Marie-Aline GERARD
Philippe WOLFF
Ronald MICURA
Jessica STEGER (PhD)
Katja FAUSTER (PhD)
Ribosome
Benoit MEYER
Guillaume BEC
Philippe WOLFF
Stefano MARZI

26. Common issues in crystallization of complexes
- a significant fraction of the protein /RNA is misfolded
- the protein concentration is incorrect
- a significant fraction of the ligand is inactive and unable to bind the protein/RNA
- the ligand concentration is incorrect
- presence of unspecific binding sites for the ligand on the protein/RNA
- the ligand do not bind the protein/RNA in experimental conditions used for crystallization
ITC can be used as a guide in protein(RNA)/ligand crystallization
Complete binding profile of protein(RNA)/ligand interaction is provided ‘for free’

27. Understanding a riboswitch using ITC

28. Total timelapse of ITC experiments: 2 days
Sample required: two large T7 transcriptions (~ 1.3 mg RNA)
Ligand
202 nt riboswitch
Problems for interpretation of chemical probing
Problems for interpretation of SPR data
Problems for crystallization of the RNA/ligand complex

29. 1st day RNA: 20µM ligand: 500µM 30.0°C 32.5°C 35.0°C 37.5°C N1 0,69
Kd1 35 nM
DH cal/mol
DS1 12,6 cal/mol/deg
N2 0,40
Kd2 193 nM
DH cal/mol
DS2 -0,310 cal/mol/deg
N1 0,58
Kd1 114 nM
DH cal/mol
DS cal/mol/deg
N2 0,57
Kd2 10 nM
DH cal/mol
DS2 5,01 cal/mol/deg
N1 0,79
Kd1 61 nM
DH cal/mol
DS1 -16,8 cal/mol/deg
N2 0,34
Kd2 NA
DH cal/mol
DS2 NA
N1 1,08
Kd1 35 nM
DH cal/mol
DS1 -27,0 cal/mol/deg

30. 2nd day RNA recycled on Centricon 10.0°C 30.0°C 6.0°C 27.0°C N1 0,69
Kd1 35 nM
DH cal/mol
DS1 12,6 cal/mol/deg
N2 0,40
Kd2 193 nM
DH cal/mol
DS2 -0,310 cal/mol/deg
N1 0,85
Kd1 61 nM
DH cal/mol
DS1 25,8 cal/mol/deg
N2 0,26
Kd2 262 nM
DH cal/mol
DS2 9,05 cal/mol/deg
N1 0,82
Kd1 NA
DH cal/mol
DS1 NA
N2 0,19
Kd2 NA
DH cal/mol
DS2 NA
N1 0,65
Kd1 NA
DH cal/mol
DS1 NA
N2 0,20
Kd2 NA
DH2 2282cal/mol
DS2 NA

31. Temperature dependance of DH for sites 1 and 2
DCp ~ kcal/mol/K for site 1
DCp ~ kcal/mol/K for site 2
DH kcal/mol
Temperature (°C)
2 alternative species with different binding properties are in solution
Site 2 is likely less folded than Site 1
Site 2 has a better affinity (Site 1 partially ‘misfolded’)

32. This Ecoli riboswitch is resistant to temperature changes
Variation of the two populations of riboswitches with the temperature
Stoichiometry
Temperature (°C)
The two populations are in equilibrium
Site 1 population is 37°C
This Ecoli riboswitch is resistant to temperature changes