Isothermal Titration Calorimetry: Principles and Experimental Design

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Isothermal titration calorimetry: Principles and experimental design

Agenda

Overview of Isothermal Titration Calorimetry ITC experimental design Data analysis Troubleshooting

2 GE Title or job number 11/2/2012 What is isothermal titration calorimetry (ITC)

A direct measurement of the heat generated or absorbed when molecules interact

Microcalorimetry offers enhanced information content

Label-free

In-solution

No molecular weight limitations

Optical clarity unimportant

Minimal or no assay development

4 GE Title or job number 11/2/2012 How Do ITCs Work?

The DP is a measured power differential between the reference and sample cells necessary to S R maintain their temperate difference at close to zero

DT DP Reference Calibration Heater Sample Calibration Heater DT~0 Cell Main Heater

5 GE Title or job number 11/2/2012 Performing an ITC experiment

Ligand in syringe Macromolecule in sample cell Syringe Heat of interaction is measured Parameters measured from a single ITC experiment: Affinity - KD

Energy (Enthalpy) - DH Reference Cell Sample Cell Number of binding sites - n

6 GE Title or job number 11/2/2012 ITC – Before titration

Ligand – in syringe Macromolecule in ITC cell

7 GE Title or job number 11/2/2012 Titration begins: First injection

Ligand in syringe Macromolecule in cell Macromolecule-ligand complex

5 As the first injection is made, all injected ligand is bound to target macromolecule.

8 GE Title or job number 11/2/2012 Return to baseline

5 The signal returns to baseline before the next injection.

9 GE Title or job number 11/2/2012 Second injection

5 As a second injection is made, again all injected ligand becomes bound to the target. 0

10 GE Title or job number 11/2/2012 Second return to baseline

5 Signal again returns to baseline before next injection.

11 GE Title or job number 11/2/2012 Injections continue

As the injections 5 continue, the target becomes saturated with ligand, so less binding occurs and the heat change starts to decrease. 0

12 GE Title or job number 11/2/2012 Injections continue

As the injections 5 continue, the target becomes saturated with ligand so less binding occurs and the heat change starts to decrease. 0

13 GE Title or job number 11/2/2012 End of titration

When the macromolecule is 5 saturated with ligand, no more binding occurs, and only heat of dilution is 0 observed.

14 GE Title or job number 11/2/2012 Experimental results

= 1/KD

15 GE Title or job number 11/2/2012 ™ MicroCal ITC systems

MicroCal™ VP-ITC MicroCal™ iTC200 MicroCal™ Auto-iTC200 • 1400 L cell • Sensitive • Unattended operation • Manual sample • Fast • Up to 75 samples/day (using single injection loading • Easy to use method) • Up to 5 • KD from mM to nM • K from mM to nM samples/day D • 200 µL cell • Sample cell is 200 µL • Upgradable to full • Easy to use automation • 96-well plate format

16 GE Title or job number 11/2/2012 Why ITC? Heat is a fundamental natural property…

A single titration can yield information on:

Overall binding affinity

Hydrogen bonds and van der Waals interactions

Hydrophobic and conformational effects

Stoichiometry calorimetry is a direct readout

18 GE Title or job number 11/2/2012 Stoichiometry

Number of ligand binding sites per macromolecule If one binding site the stoichiometry is 1 By convention a “Ligand” has one binding site A “Macromolecule” can have more than one binding site

19 GE Title or job number 11/2/2012 Effective Binding Affinity Range

KD in mM to nM range

Weak binding – low C-value method

Tight binding - minimize injection volume and concentration or use competitive (displacement) binding procedure and fitting model

20 GE Title or job number 11/2/2012 Thermodynamics

KB – binding constant

[L] x [M] K = 1/ K = D B [ML]

DG = RT lnKD

DG = DH - T DS

` 21 GE Title or job number 11/2/2012

Free energy change

 DG is change in free energy

 DG  0 for spontaneous process

 More negative DG, higher affinity

22 GE Title or job number 11/2/2012 Enthalpy change

 DH – measure of the energy content of the bonds broken and created. The dominant contribution is from hydrogen bonds.  Negative value indicates enthalpy change favoring the binding  Solvents play a role

23 GE Title or job number 11/2/2012 Entropy change

 DS – positive for entropically driven reactions  Hydrophobic interactions  Solvation entropy (favorable) due to release of water  Conformational degrees of freedom (unfavorable)

24 GE Title or job number 11/2/2012 Microcalorimetry provides a total picture of binding energetics

Overall binding affinity KD correlates with IC50 or EC50. This is directly related to ∆G, the total free binding energy

∆H, enthalpy is indication of changes in hydrogen and van der Waals bonding -T∆S -T∆S, entropy is indication of changes ∆H in hydrophobic interaction and conformational changes N, stoichiometry indicates the ratio of ligand-to-macromolecule binding DG = DH -TDS

25 GE Title or job number 11/2/2012 Same affinity, different energetics! All three interactions have the same binding energy (∆G) Unfavorable

10 A. Good hydrogen bonding with 5 unfavorable conformational 0 change ∆G -5 ∆H

kcal/mole -T∆S B. Binding dominated by -10

hydrophobic interaction -15

-20 C. Favorable hydrogen bonds A B C and hydrophobic interaction Favorable

ITC results are used to get insights into mechanism of binding

26 GE Title or job number 11/2/2012 How to get good ITC data C Values [M] 0 C = K -2 D

-4 Example: 0.05 -6 0.5 -8 Kd = 100nM

kcal/mole of injectant of kcal/mole 5 -10 50 -12 [M] = 100nM, C=1 -14 500 [M] = 5uM, C=50

-16 0.0 0.5 1.0 1.5 2.0 Molar Ratio [M]:[L] – 1:10 for n=1

28 GE Title or job number 11/2/2012

C Values tim e (m in) 0 30 60 90 120 150

-1

(a) µcal/s -2

-4 c = 5 -8 Kd = 1.2 µM (b) -12 c = 40

-16

kcal/mol of injected Ras 0.0 0.5 1.0 1.5 2.0 2.5 3.0 molar ratio (Ras / RalGDS-RBD) 29 GE Title or job number 11/2/2012 C Values in ITC

C = {[M]tot / KD} * N

C = 20-100 very good C = 10-500 good

C = 1-5 and 500-1000 OK

C = < 1 and > 1000 not wanted

30 GE Title or job number 11/2/2012 ITC experimental design

High C Low C

0 00 0

-2 -2-2 10< [Protein]/K <100 -0.2 [Protein]/K >> 1000 D

of of injectant D

1 Fitted: N, KD, DH

- Fitted: N, DH

[Protein]/KD < 1

kcal/moleof injectant N fixed -4-4 -0.4

-4 Fitted: KD, DH kcal kcal mol

0.0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 4 8 12 16 Molar Ratio Molar ratio

31 GE Title or job number 11/2/2012 ITC experimental design

KD (Biacore) µM [Protein] µM [Compound] µM [Protein] / KD

<0.5 10 100 >20

0.5-2 30 300 15-60

2-10 50 500 5-25

10-100 30 40*K 0.3-3 D Fixed stoichiometry >100 30 20*KD <0.3

34 GE Title or job number 11/2/2012 Low C Experiments

Extending the applications of ITC – E.g. Fragment Based Drug Discovery

Saving Protein

35 GE Title or job number 11/2/2012 C Values-Low

0 -1.1

-2 -1.2

-4

-1.3 -6

-1.4

-8 kcal/mole of injectant of kcal/mole kcal/mole of injectant of kcal/mole -1.5 -10

-12 -1.6

-14 -1.7

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0 2 Molar Ratio Molar Ratio

Note different scales 36 GE Title or job number 11/2/2012 C Values-Low

0 Increase ligand concentration but not

the ‘valuable’ protein kcal/mole of injectant of kcal/mole

-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26

Molar Ratio 37 GE Title or job number 0------>26 11/2/2012 Competition Experiments

Extend the affinity range that ITC can be used • Submillimolar (10-2) to picomolar (10-12)

38 GE Title or job number 11/2/2012 Competition Experiments

0 High C Experiment -2 Poor affinity estimates -4

-6 100% Binding 0% Binding

-8 kcal/mole of injectant of kcal/mole

-10

-12

-14

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio 39 GE Title or job number 11/2/2012 Competitive (displacement) ITC

Used to extend range of KB determined by ITC Tight binding ligand A and weak binding ligand B bind to same site on macromolecule 1st experiment: Ligand B titrated into macromolecule. Determine KB and DH 2nd experiment: Macromolecule + ligand B in cell, titrated with Ligand A. Ligand A displaces ligand B. Use displacement model for data analysis

40 GE Title or job number 11/2/2012 Tight Binders

Protein Tight Ligand Competitor

0 0

-2 -2 -4 -4 Software -6 -6

-8 -8

kcal/mole of injectant of kcal/mole -10

kcal/mole of injectant of kcal/mole can ‘pull out’ the K -10 D

-12 -12

-14 of the tight one -14

-16 -16 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio Molar Ratio

41 GE Title or job number 11/2/2012 Weak Binders

-2

-4 +

-6

-8

kcal/mole of injectant of kcal/mole -10

-12

-14 0 -16

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

Molar Ratio kcal/mole of injectant of kcal/mole -10

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio

alone kcal/mole of injectant of kcal/mole -10

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio Software can pull the

Weak KD out

42 GE Title or job number 11/2/2012 Competition Experimental Design

C = [cell]/KD,app

KD,app= (1/KD,S)/(1+1/KD,W[W])

Where KD,S and KD,W are the affinity of the strong and weak binders respectively and W is the concentration of the weak binder

43 GE Title or job number 11/2/2012 Displacement ITC – HIV-1 Protease- Inhibitor Binding

Amprenavir Acetyl pepstatin Amprenavir + acetyl pepstatin

Unable to determine KB KB of 3.1 x 1010 M-1

Ohtaka, et al, Protein Sci. 11, 1908-1916 (2002) 46 GE Title or job number 11/2/2012 ITC practical considerations ITC practical considerations

3.55 -2

3.50 protein A in syringe titrated into buffer -4 3.45

3.40 -6

3.35 -8

µcal/sec 3.30 protein A in syringe titrated into protein B

3.25 -10 kcal/mole of injectant of kcal/mole

3.20 -12

3.15 0.00 10.00 20.00 30.00 40.00 50.00 -14 Time (min)

-16

-18 0 1 2 3 Molar Ratio 54 GE Title or job number 11/2/2012 Good Experimental Design

Use the correct ‘C’ value

C = [M]tot.n / KD

C = 20-100 very good C = 10-500 good

C = 1-5 and 500-1000 OK

C < 1 and > 1000 not wanted

56 GE Title or job number 11/2/2012 Ligand concentration

[L] = 10 to 20 x [M] [L] - Minimum 50 M May need to be adjusted based on experiment At end of ITC, for N of 1, final [L]/[M] ratio should be 2 to 4 to ensure saturation of all M binding sites

60 GE Title or job number 11/2/2012 iTC200 Experimental Design Tab

61 GE Title or job number 11/2/2012 With little prior knowledge

Good Starting Conditions

100M Ligand in the Syringe and 10M Macromolecule in the cell 16 x 2.5 l injections

Detect KDs of 10 M to 10 nM

Ideal for KDs of 2 M 100 nM

62 GE Title or job number 11/2/2012 Sample preparation

Use dialysis or buffer exchange column

Check calibration pipettes-by weight

Retain the dialysate/exchanged buffer

Adopt and stick to a reproducible protocol

63 GE Title or job number 11/2/2012 ITC – Choice of Buffer

What buffer(s) are protein and ligand stable in? What pH? Buffers used for other binding studies Solubility Requirements of additives for binding, solubility or stability •Salt •Detergent •Reducing agent •DMSO •Other

64 GE Title or job number 11/2/2012 Choice of buffer Buffers have ionization enthalpies:

BH B- + H+ DH ion

Use buffers with DHion ~ 0 Including; phosphate, acetate, formate, citrate, sulfate, cacodylate, glycine

Quaternary amines (e.g. Tris) have high DHion

66 GE Title or job number 11/2/2012 Choice of buffer

Avoid DTT

Unstable and undergoes oxidation

High background heat

Use b-mercaptoethanol & TCEP

TCEP is not stable in phosphate buffer

Use conditions in which your protein is ‘happy’

67 GE Title or job number 11/2/2012 Sample preparation: small molecule ligand If ligand is too small to dialyze, be sure material is desalted prior to final preparation Use final dialysis buffer of macromolecule to dissolve ligand Match pH

70 GE Title or job number 11/2/2012 ITC – “Reverse Titration”

Can also have ligand in cell – lower concentration requirement. Have macromolecule in syringe at appropriate (higher) concentration, Need to be sure can have protein at the appropriate concentration. At saturation: no more free ligand in ITC cell If other than 1:1 binding, use “ligand in cell” option for curve fitting. Different model used.

71 GE Title or job number 11/2/2012 iTC200 Experimental design Injection volume and duration • 0.5 to 3 l ((range 0.1-38 l) • Injection rate is 0.5 l/sec • Spacing • Typical 120 secs-may want to extend to 180 seconds or more if using no feedback with large heats • Be sure baseline returns before next injection • If this does not occur, increase time between injections Filter period • 5 sec or less recommended • Can be increased for slow reactions

72 GE Title or job number 11/2/2012 iTC200 Experimental design

Typical temperature: 25 °C Number of injections: 12-18 Reference power • Instrument baseline • Set 5 cal/sec • If very exothermic, increase setting so DP does not go below zero Initial delay • 60 sec minimum • Establish baseline before 1st injection

73 GE Title or job number 11/2/2012 iTC200 Experimental design

Feedback mode

• High feedback for most ITC experiments • Low or No feedback will give better S/N but will take longer time (increase time between injections) – normally used when working with small heats Stirring speed • 1000 rpm (1500 for SIM)

74 GE Title or job number 11/2/2012 Auto- iTC200

Macromolecule/protein (for cell): Need 400 l in 96 well plate Ligand (for syringe): Need 120 l in 96 well plate Set appropriate scripts for cell/syringe filling and cleaning

75 GE Title or job number 11/2/2012 ITC – Enthalpy Changes

DHobserved by ITC is total of :

DHbinding

DHionization

DHconformation Any non-specific effects (buffer mismatch, pH mismatch, heat of dilution, heat of ligand dissociation) Need to account for these effects by appropriate controls and experimental conditions

76 GE Title or job number 11/2/2012 Controls Injection of syringe material into buffer-

4.75 4.70 A into Buffer 4.65 4.60

4.55 A into B 4.50

µcal/sec 4.45

4.40

4.35

4.30

4.25 0 10 20 30 40 50 60 70 80 90 Time (min) Peaks should be similar in magnitude to those at the end of the actual titration experiment and constant

77 GE Title or job number 11/2/2012 Controls

If they are constant- subtract average peak size from the experimental data (Using the ‘Math’ command)

78 GE Title or job number 11/2/2012 Controls

If they are not constant Buffer Mismatch –e.g. solvent component missing Dissociation of the syringe material upon dilution into buffer- impact on apparent KD Genuine change in heat of dilution with concentration.

79 GE Title or job number 11/2/2012 Buffer Mismatch

7 2% DMSO into A: Matched solution: both cell & syringe have same solution (280 l DMSO added to 14 ml buffer).

2% DMSO B: Slightly mismatched solution: syringe: 20 l DMSO added to 1.0 ml buffer; 6 cell: 280 l DMSO added to 14 ml buffer.

2% DMSO into 5 C: 2 % mismatch in DMSO: syringe: 20 l DMSO added to 1.0 ml buffer; cell: buffer only (no DMSO). 1.95% DMSO 4

3 µcal/sec

2% DMSO into 2 0% DMSO

0 -5 0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

80 GE Title or job number 11/2/2012 Dissociation of Syringe Material into Buffer

Calorimetric dilution data showing the effects of different ligands on dilution of insulin

Ref: Lovatt M, Cooper A and Camillerri P (1996) Eur. Biophys. J. 24:354-357

81 GE Title or job number 11/2/2012 Changing Heats of Dilution

The control experiment should be fit to a straight line – (or geometric function (if required- Caution!)

This best fit line can then be subtracted from titration experiment before fitting to the appropriate model.

82 GE Title or job number 11/2/2012 For Each ITC Experiment

Start with clean cell and syringe Prepare macromolecule and ligand in matched buffer Perform control titration(s) to establish heat of dilution Set appropriate scan parameters to generate full binding isotherm

86 GE Title or job number 11/2/2012 Troubleshooting Bent Syringe

DP 0

DP 107 GE Title or job number 11/2/2012 Syringe Height iTC200 - Syringe Holding Nut Loose

DP 0

DP 108 GE Title or job number 11/2/2012 Baseline Position/Drift

Baseline position is the first diagnostic for data quality-information on • Cell cleanliness • ‘Sticky’ proteins • Air Bubbles • Time between injections

109 GE Title or job number 11/2/2012 Bubbles

DP 0

DP 110 GE Title or job number 11/2/2012 Not long enough between injections

16 µcal/sec

14 0.00 33.33 66.67 100.00 133.33 166.67 Time (min)

111 GE Title or job number 11/2/2012 Not long enough between injections

8 µcal/sec

2 -8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 66.67 Time (min)

112 GE Title or job number 11/2/2012 ‘Sticky’ Proteins or Cleanliness

Change in baseline

2 µcal/sec

-33.33 0.00 33.33 66.67 100.00 133.33 166.67 200.00 233.33 266.67 Time (min)

113 GE Title or job number 11/2/2012 ‘Sticky’ Proteins or Cleanliness

0 µcal/sec

-2

-4 0.00 8.33 16.67 25.00 33.33 41.67 50.00 Time (min)

114 GE Title or job number 11/2/2012 ‘Sticky’ Proteins or Cleanliness

16 µcal/sec

-16.67 0.00 16.67 33.33 50.00 66.67 83.33 100.00 116.67 133.33 Time (min)

115 GE Title or job number 11/2/2012 Non sigmoidal binding isotherm

No Binding No Heat Buffer mismatch More than one binding event

116 GE Title or job number 11/2/2012 Buffer Mismatch-No Dialysis

2.5

2.0

1.5

1.0 µcal/sec

0.5

without dialysis 0.0 with dialysis

-0.5 0 20 40 60 80 100 120 140 160 180 Time (min)

117 GE Title or job number 11/2/2012 Effect of DMSO Mismatch

7 2% DMSO into A: Matched solution: both cell & syringe have same solution (280 l DMSO added to 14 ml buffer).

2% DMSO-same stock B: Slightly mismatched solution: syringe: 20 l DMSO added to 1.0 ml buffer; 6 cell: 280 l DMSO added to 14 ml buffer.

2% DMSO into 5 C: 2 % mismatch in DMSO: syringe: 20 l DMSO added to 1.0 ml buffer; cell: buffer only (no DMSO). 2% DMSO-separate stocks 4

3 µcal/sec

2% DMSO into 2 0% DMSO

0 -5 0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

118 GE Title or job number 11/2/2012 Ligand preparation from DMSO

5 mM ligand Dialysate in 100% DMSO 50 l 950 l buffer

250 M ligand in 5 % DMSO

119 GE Title or job number 11/2/2012 Match DMSO in the protein solution

DMSO 25 M dialyzed 50 l 950 l protein

1 mL of 23.75 M protein in 5 % DMSO

120 GE Title or job number 11/2/2012 The Cure

Dialyze or use desalting column Check for additives that are not in both cell and syringe- Also-ask what was sample purified from e.g. was protein lyophilized in buffer and not dialyzed Check pH of final solutions-should differ by less than 0.1 pH units. This issue is common when working with high concentrations of ligand-e.g. 500 M and above-weak binding

121 GE Title or job number 11/2/2012 ITC: low heat

4.52 Control: ligand into buffer 4.50 4.48 4.46 4.44 Heats for experiment 4.42 4.40 same as control 4.38

4.36 µcal/sec 4.34 4.32 ExperimentL ligand into protein 4.30 4.28 4.26

0.00 16.67 33.33 50.00 66.67 83.33 Time (min) 125 GE Title or job number 11/2/2012 The Cure

Change experimental temperature by at least 10 °C AND/OR Increase sample concentration

126 GE Title or job number 11/2/2012 Unexpected Stoichiometry

19.3

19.2

19.1

19.0

18.9 µcal/sec

18.8

18.7

18.6 0.00 33.33 66.67 100.00 133.33 166.67 Time (min)

127 GE Title or job number 11/2/2012 Instrument reference power too low

Oscillating signal: 2 Power below 0

µcal/sec -2

-4

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 Time (min) 128 GE Title or job number 11/2/2012 Insoluble Ligands

‘One site’ interactions are symmetrical and as such the ligand can be put in the cell and the protein in the syringe

129 GE Title or job number 11/2/2012 Cure-Reverse the Titration

Time (min) -10 0 10 20 30 40 50 60 70 80 90 100 110 120

29.5µM Protein titrated with 0.0

1.1mM Compound -0.5

-1.0 µcal/sec -1.5 11.5µM Compound titrated with 2 0 179µM Protein -2 -4 -6 -8 -10

Time (min) -12 kcal/moleof injectant -10 0 10 20 30 40 50 60 70 80 90 100 0.0 0.5 1.0 1.5 2.0 2.5 Molar Ratio 0.0

-0.2

-0.4 Parameter Ligand in syringe Ligand in cell µcal/sec -0.6 n 0.99 0.97 -0.82 0 -2 K d 104 nM 105 nM -4 -6 DG o -9.4 kcal/mol -9.4 kcal/mol -8 -10 DHo -11.9 kcal/mol -12.4 kcal/mol -12 -14 kcal/moleof injectant o 0.0 0.5 1.0 1.5 2.0 2.5 TD S -2.5 kcal/mol -3.0 kcal/mol Molar Ratio 130 GE Title or job number 11/2/2012 Stoichiometry

“N” is the average number of binding sites per mole of protein in your solution, assuming: • that all binding sites are identical and independent • that you have pure protein (and ligand) • that you have given the correct protein and ligand concentrations • that all your protein is active

131 GE Title or job number 11/2/2012 ITC Maintenance

Cell cleaning: Water/buffer 20 % Contrad 70 Keep water in cell when not in use Reference cell: Replace water once a week

140 GE Title or job number 11/2/2012 Thank you!

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141 GE Title or job number 11/2/2012