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Isothermal titration calorimetry: Principles and experimental design
2 GE Title or job number
11/2/2012
Agenda
Overview of Isothermal Titration Calorimetry
ITC experimental design
Data analysis
Troubleshooting
What is isothermal titration calorimetry (ITC)
A direct measurement of the heat generated or absorbed when molecules interact
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Microcalorimetry offers enhanced information content
Label-free
In-solution
No molecular weight limitations
Optical clarity unimportant
Minimal or no assay development
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How Do ITCs Work?
Reference Calibration Heater
Cell Main Heater
Sample Calibration Heater
DP
DT
S R The DP is a measured power differential between the reference and sample cells necessary to maintain their temperate difference at close to zero
DT~0
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Performing an ITC experiment Ligand in syringe
Macromolecule in sample cell
Heat of interaction is measured
Parameters measured from a single ITC experiment:
Affinity - KD
Energy (Enthalpy) - DH
Number of binding sites - n
Reference Cell Sample Cell
Syringe
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Ligand – in syringe Macromolecule in ITC cell
ITC – Before titration
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Ligand in syringe
Macromolecule in cell
Macromolecule-ligand complex
As the first injection is made, all injected ligand is bound to target macromolecule.
Titration begins: First injection
5
0
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The signal returns to baseline before the next injection.
Return to baseline
5
0
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As a second injection is made, again all injected ligand becomes bound to the target.
Second injection
5
0
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Second return to baseline
Signal again returns to baseline before next injection.
5
0
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As the injections continue, the target becomes saturated with ligand, so less binding occurs and the heat change starts to decrease.
Injections continue
5
0
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As the injections continue, the target becomes saturated with ligand so less binding occurs and the heat change starts to decrease.
Injections continue
5
0
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When the macromolecule is saturated with ligand, no more binding occurs, and only heat of dilution is observed.
End of titration
5
0
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Experimental results
= 1/KD
5
0
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MicroCal™ VP-ITC MicroCal™ iTC200 MicroCal™ Auto-iTC200
• 1400 L cell
• Manual sample loading
• Up to 5 samples/day
• Sensitive
• Fast
• Easy to use
• KD from mM to nM
• 200 µL cell
• Upgradable to full automation
• Unattended operation
• Up to 75 samples/day (using single injection method)
• KD from mM to nM
• Sample cell is 200 µL
• Easy to use
• 96-well plate format
MicroCal™ ITC systems
Why ITC?
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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
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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
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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
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Thermodynamics
KB – binding constant
KD = 1/ KB =
DG = RT lnKD
DG = DH - T DS
`
[L] x [M]
[ML]
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Free energy change
DG is change in free energy
DG 0 for spontaneous process
More negative DG, higher affinity
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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
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Entropy change
DS – positive for entropically driven reactions
Hydrophobic interactions
Solvation entropy (favorable) due to release of water
Conformational degrees of freedom (unfavorable)
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Microcalorimetry provides a total picture of binding energetics
∆H
-T∆S
Overall binding affinity KD correlates with IC50 or EC50. This is directly related to ∆G, the total free binding energy
DG = DH -TDS
∆H, enthalpy is indication of changes in hydrogen and van der Waals bonding
-T∆S, entropy is indication of changes in hydrophobic interaction and conformational changes
N, stoichiometry indicates the ratio of ligand-to-macromolecule binding
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Same affinity, different energetics! All three interactions have the same binding energy (∆G) A. Good hydrogen bonding with
unfavorable conformational
change
B. Binding dominated by
hydrophobic interaction
C. Favorable hydrogen bonds
and hydrophobic interaction
ITC results are used to get insights into mechanism of binding
-20
-15
-10
-5
0
5
10
kc
al/
mo
le ∆G
∆H
-T∆S
Favorable
Unfavorable
A B C
How to get good ITC data
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C Values
0.0 0.5 1.0 1.5 2.0
-16
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
0.05
0.5
5
50
500
C = [M]
KD
Example: Kd = 100nM [M] = 100nM, C=1 [M] = 5uM, C=50 [M]:[L] – 1:10 for n=1
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-2
-1
0
0 30 60 90 120 150
(a)
tim e (m in)
µc
al/
s
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-16
-12
-8
-4
0
(b)
molar ratio (Ras / RalGDS-RBD)
kc
al/
mo
l o
f in
jec
ted
Ra
s
c = 5
c = 40
Kd = 1.2 µM
C Values
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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
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ITC experimental design kc
al
mo
l-1 o
f in
jec
tan
t
Molar ratio
-4
-2
0
0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0
-4
-2
0
Molar Ratio
kca
l/m
ole
of in
ject
ant
10< [Protein]/KD <100
Fitted: N, KD, DH
0.0 0.5 1.0 1.5 2.0
-4
-2
0
Molar Ratio
kca
l/m
ole
of in
ject
ant
-4
-2
0
0 0.5 1.0 1.5 2.0
[Protein]/KD >> 1000
Fitted: N, DH
0.0 0.5 1.0 1.5 2.0
-4
-2
0
Molar Ratio
kca
l/m
ole
of in
ject
ant
-0.4
-0.2
0
0 4 8 12 16
[Protein]/KD < 1 N fixed
Fitted: KD, DH
High C Low C
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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*KD 0.3-3
>100 30 20*KD <0.3
Fixed stoichiometry
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Low C Experiments
Extending the applications of ITC
– E.g. Fragment Based Drug Discovery
Saving Protein
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C Values-Low
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
0 2
-1.7
-1.6
-1.5
-1.4
-1.3
-1.2
-1.1
Molar Ratio
kcal/m
ole
of in
jecta
nt
Note different scales
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C Values-Low
Increase ligand concentration but not the ‘valuable’ protein
0 2 4 6 8 10 12 14 16 18 20 22 24 26
-2
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
0--------------->26
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Competition Experiments
Extend the affinity range that ITC can be used
• Submillimolar (10-2) to picomolar (10-12)
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Competition Experiments
High C Experiment
Poor affinity estimates
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
100% Binding 0% Binding
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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
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Tight Binders
Protein
Tight Ligand
Competitor
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-16
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-16
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of in
jecta
nt Software
can ‘pull out’ the KD
of the tight one
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Weak Binders
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-16
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-10
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-10
0
Molar Ratio
kcal/m
ole
of in
jecta
nt
alone
+
Software can pull the Weak KD out
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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
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Displacement ITC – HIV-1 Protease- Inhibitor Binding
Amprenavir Acetyl pepstatin Amprenavir + acetyl pepstatin
Ohtaka, et al, Protein Sci. 11, 1908-1916 (2002)
Unable to determine KB KB of 3.1 x 1010 M-1
ITC practical considerations
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ITC practical considerations
0.00 10.00 20.00 30.00 40.00 50.00
3.15
3.20
3.25
3.30
3.35
3.40
3.45
3.50
3.55
protein A in syringe titrated into protein B
Time (min)
µca
l/se
c
protein A in syringe titrated into buffer
0 1 2 3
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
2
Molar Ratio
kcal/m
ole
of in
jecta
nt
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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
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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
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iTC200 Experimental Design Tab
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With little prior knowledge
Good Starting Conditions
100M Ligand in the Syringe and
10M 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
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Use dialysis or buffer exchange column
Check calibration pipettes-by weight
Retain the dialysate/exchanged buffer
Adopt and stick to a reproducible protocol
Sample preparation
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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
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Choice of buffer
Buffers have ionization enthalpies:
BH B- + H+
Use buffers with DHion ~ 0
Including; phosphate, acetate, formate, citrate, sulfate, cacodylate, glycine
Quaternary amines (e.g. Tris) have high DHion
DHion
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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’
Choice of buffer
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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
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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.
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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
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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
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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)
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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
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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
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Controls Injection of syringe material into buffer-
Peaks should be similar in magnitude to those at the end of the actual titration experiment and constant
0 10 20 30 40 50 60 70 80 90
4.25
4.30
4.35
4.40
4.45
4.50
4.55
4.60
4.65
4.70
4.75
A into B
A into Buffer
Time (min)
µca
l/se
c
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Controls
If they are constant-
subtract average peak size from the experimental data (Using the ‘Math’ command)
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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.
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Buffer Mismatch
-5 0 5 10 15 20 25 30 35 40 45 50 55
0
1
2
3
4
5
6
7
C: 2 % mismatch in DMSO: syringe: 20 l DMSO added to 1.0 ml buffer; cell: buffer only (no DMSO).
B: Slightly mismatched solution: syringe: 20 l DMSO added to 1.0 ml buffer;
cell: 280 l DMSO added to 14 ml buffer.
A: Matched solution: both cell & syringe have same solution (280 l DMSO added to 14 ml buffer).
Time (min)
µcal/sec
2% DMSO into 2% DMSO
2% DMSO into 1.95% DMSO
2% DMSO into 0% DMSO
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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
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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.
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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
Troubleshooting
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Bent Syringe
0
5
DP
DP
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Syringe Height
0
5
DP
iTC200 - Syringe Holding Nut Loose
DP
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Baseline Position/Drift
Baseline position is the first diagnostic for data quality-information on
• Cell cleanliness
• ‘Sticky’ proteins
• Air Bubbles
• Time between injections
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Bubbles
0
5
DP
DP
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Not long enough between injections
0.00 33.33 66.67 100.00 133.33 166.67
14
15
16
17
18
19
Time (min)
µca
l/se
c
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Not long enough between injections
-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 66.67
2
4
6
8
10
12
14
16
Time (min)
µca
l/se
c
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‘Sticky’ Proteins or Cleanliness
-33.33 0.00 33.33 66.67 100.00 133.33 166.67 200.00 233.33 266.67
0
2
4
Time (min)
µca
l/se
c Change in baseline
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‘Sticky’ Proteins or Cleanliness
0.00 8.33 16.67 25.00 33.33 41.67 50.00
-4
-2
0
2
4
6
Time (min)
µca
l/se
c
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‘Sticky’ Proteins or Cleanliness
-16.67 0.00 16.67 33.33 50.00 66.67 83.33 100.00 116.67 133.33
14
16
Time (min)
µca
l/se
c
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Non sigmoidal binding isotherm
No Binding
No Heat
Buffer mismatch
More than one binding event
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Buffer Mismatch-No Dialysis
0 20 40 60 80 100 120 140 160 180
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
without dialysis
with dialysis
Time (min)
µca
l/se
c
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Effect of DMSO Mismatch
-5 0 5 10 15 20 25 30 35 40 45 50 55
0
1
2
3
4
5
6
7
C: 2 % mismatch in DMSO: syringe: 20 l DMSO added to 1.0 ml buffer; cell: buffer only (no DMSO).
B: Slightly mismatched solution: syringe: 20 l DMSO added to 1.0 ml buffer;
cell: 280 l DMSO added to 14 ml buffer.
A: Matched solution: both cell & syringe have same solution (280 l DMSO added to 14 ml buffer).
Time (min)
µcal/sec
2% DMSO into 2% DMSO-same stock
2% DMSO into 2% DMSO-separate stocks
2% DMSO into 0% DMSO
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Ligand preparation from DMSO
5 mM ligand in 100% DMSO 50 l
Dialysate buffer 950 l
250 M ligand in 5 % DMSO
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Match DMSO in the protein solution
DMSO 50 l
25 M dialyzed protein 950 l
1 mL of 23.75 M protein in 5 % DMSO
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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
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ITC: low heat
0.00 16.67 33.33 50.00 66.67 83.33
4.26
4.28
4.30
4.32
4.34
4.36
4.38
4.40
4.42
4.44
4.46
4.48
4.50
4.52
Time (min)
µca
l/se
c
Control: ligand into buffer
ExperimentL ligand into protein
Heats for experiment same as control
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The Cure
Change experimental temperature by at least 10 °C
AND/OR
Increase sample concentration
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Unexpected Stoichiometry
0.00 33.33 66.67 100.00 133.33 166.67
18.6
18.7
18.8
18.9
19.0
19.1
19.2
19.3
Time (min)
µca
l/se
c
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Instrument reference power too low
-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33
-4
-2
0
2
Time (min)
µca
l/se
c
Oscillating signal: Power below 0
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Insoluble Ligands
‘One site’ interactions are symmetrical and as such the ligand can be put in the cell and the protein in the syringe
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-1.5
-1.0
-0.5
0.0
-10 0 10 20 30 40 50 60 70 80 90 100 110 120
Time (min)
µca
l/se
c
0.0 0.5 1.0 1.5 2.0 2.5
-12
-10
-8
-6
-4
-2
0
2
Molar Ratio
kca
l/m
ole
of in
jecta
nt
-0.8
-0.6
-0.4
-0.2
0.0
-10 0 10 20 30 40 50 60 70 80 90 100
Time (min)
µca
l/se
c
0.0 0.5 1.0 1.5 2.0 2.5
-14
-12
-10
-8
-6
-4
-2
0
2
Molar Ratio
kca
l/m
ole
of in
jecta
nt
Parameter
Ligand in syringe
Ligand in cell
n
0.99
0.97
Kd
104 nM
105 nM
DGo
-9.4 kcal/mol
-9.4 kcal/mol
DHo
-11.9 kcal/mol
-12.4 kcal/mol
TDSo
-2.5 kcal/mol
-3.0 kcal/mol
29.5µM Protein titrated with 1.1mM Compound
11.5µM Compound titrated with 179µM Protein
Cure-Reverse the Titration
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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
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ITC Maintenance
Cell cleaning:
Water/buffer
20 % Contrad 70
Keep water in cell when not in use
Reference cell:
Replace water once a week
141 GE Title or job number
11/2/2012
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