Isothermal titration calorimetry:

Principles and experimental design

Stoyan Milev, Ph.D

GE Healthcare

stoyan.milev@ge.com
Agenda

Overview of Isothermal Titration Calorimetry

ITC experimental design
Data analysis
Troubleshooting

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What is isothermal titration
calorimetry (ITC)
A direct measurement of the heat generated or
absorbed when molecules interact
ITC applications
Study interactions between:
Protein-small molecule
Enzyme-inhibitor
Protein-protein
Protein-DNA
Protein-lipid
Protein-carbohydrate
Etc.

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Microcalorimetry offers enhanced
information content
Experimental biological relevance
Label-free
In-solution
No molecular weight limitations
Optical clarity unimportant
Minimal assay development

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 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
Cell Main Heater
DT~0
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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

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ITC Before titration

Ligand in syringe
Macromolecule in ITC cell

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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.

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Return to baseline

5 The signal returns

to baseline before
the next injection.

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Second injection

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

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Second return to baseline

5
Signal again
returns to baseline
before next
injection.

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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

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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

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End of titration

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

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Experimental results

= 1/KD

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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
loading (using single injection
Easy to use
method)
Up to 5 KD from mM to nM KD from mM to nM
samples/day
200 L cell Sample cell is 200 L
Upgradable to full Easy to use
automation 96-well plate format

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Why ITC?
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

[L] x [M]
KD = 1/ KB =
[ML]

DG = RT lnKD

DG = DH - T DS
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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
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
-TS
bonding
H
-TS, entropy is indication of changes
in hydrophobic interaction and
conformational changes
N, stoichiometry indicates the ratio of
ligand-to-macromolecule binding
DG = DH -TDS

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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

kcal/mole
-5 H
-TS
B. Binding dominated by -10

hydrophobic interaction -15

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

ITC results are used to get insights into mechanism of binding

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How to get good ITC data
C Values
[M]
0 C=
-2
KD

Example:
-4

0.05
kcal/mole of injectant

-6

0.5
-8
Kd = 100nM
-10 5
50
-12
[M] = 100nM, C=1
500
-14
[M] = 5uM, C=50
-16

0.0 0.5 1.0 1.5 2.0

Molar Ratio
[M]:[L] 1:10 for n=1
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C Values 0 30 60
tim e (m in)
90 120 150

0
cal/s

-1
(a)
-2
kcal/m ol of injected Ras

-4
c=5
-8 Kd = 1.2 M

c = 40 (b)
-12

-16

0.0 0.5 1.0 1.5 2.0 2.5 3.0

m olar ratio (R as / R alG D S -R B D )

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C Values in ITC

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

C = 10-100 very good

C = 5-500 good

C = 1-5 and 500-1000 OK

C = < 1 and > 1000 not wanted

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 ITC experimental design

High C Low C

00 00 00
kcal mol-1 of injectant

kcal/mole of injectant
kcal/mole of injectant
kcal/mole of injectant

-2-2 -2-2 10< [Protein]/KD <100 -0.2-2

[Protein]/KD >> 1000
Fitted: N, DH Fitted: N, KD, DH
[Protein]/KD < 1
N fixed
-4-4 -4-4 -0.4-4 Fitted: KD, DH

0.0 0.5 1.0 1.5 2.0

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

Molar ratio

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ITC experimental design

KD (Biacore) M [Protein] M [Compound] M [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 (Biacore) 0.3-3

Fixed
>100 30 20*KD (Biacore) <0.3
stoichiometry

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 C Values-Low
0
-1.1

-2
-1.2

-4

-1.3

kcal/mole of injectant
kcal/mole of injectant

-6

-1.4
-8

-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 37

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 C Values-Low
0
Increase ligand
concentration but not
the valuable protein
kcal/mole of injectant

-2
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Molar Ratio 38
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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)
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ITC practical considerations
 ITC practical considerations
2

3.55
-2
3.50

protein A in syringe titrated into buffer -4

3.45

kcal/mole of injectant
3.40 -6
cal/sec

3.35
-8
3.30
protein A in syringe titrated into protein B
3.25 -10

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 56
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MicroCal iTC200:
Minimum Heat=Minimum [Protein]
For iTC200:
Average heat required per injection ~ 1 cal
Need ~ 10 injections
Average DH of binding is ~ -5 kcal/mol
Therefore minimum sample concentration, [Prot], required in a 200 l cell is

Total heat, Q, = 1 cal/injection * 10 injections = 10 cal

Q = (cell volume) x (DH) x [Prot]

[Prot] = (10 cal)/(200 l)/(5,000 cal/moles)]

Min [Prot] ~ 10 M 59
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Ligand concentration

[L] = 10 to 20 x [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

300M Ligand in the Syringe and

30M Macromolecule in the cell
16 x 2.5 l injections

Ideal for KDs of 1 M 100 nM

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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

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Choice of buffer
Buffers have ionization enthalpies:

BH B- + H+
DHion

Use buffers with DHion ~ 0

Including; phosphate, acetate, formate, citrate,
sulfate, cacodylate, glycine
Quaternary amines (e.g. Tris) have high DHion
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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

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Buffer Mismatch-No Dialysis

2.5

2.0

1.5
cal/sec

1.0

0.5

without dialysis
0.0
with dialysis

-0.5
0 20 40 60 80 100 120 140 160 180

Time (min)
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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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iTC200 Experimental design

Injection volume recommended 1 to 3 l ((range 0.1-38 l)

Injection rate should be 0.5 l/sec
Spacing- typical 120 to 180 sec
Filter period - 5 sec or less recommended
Reference power typical 6 ucal/sec
High feedback for most ITC experiments
Stirring - 1000 rpm

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iTC200

Macromolecule (in the cell):

Need 300 l

Ligand (in the syringe):

Need 70 l

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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-
4.75

4.70
A into Buffer
4.65

4.60

4.55
cal/sec

A into B
4.50

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
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Buffer Mismatch

2% DMSO into 7
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

cal/sec
3

2% DMSO into 2
0% DMSO
1

0
-5 0 5 10 15 20 25 30 35 40 45 50 55

Time (min)

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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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Troubleshooting
Why Do ITC Experiments Not Work
as Expected?
Concentration errors because KD is unknown or
different value than expected
Too little heat change
Never reach saturation
Rapidly saturate binding sites
Incorrect concentrations used
Buffer mismatch too large heat of dilution
Other sources of heat change in system
No binding
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The Symptoms

Baseline Position
Baseline Drift
Split (oscillating) peaks
Non sigmoidal binding isotherm
Stoichiometry far from 1 or integer value

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Bent Syringe

DP
0

DP 113
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 Syringe Height
iTC200 - Syringe Holding Nut Loose

DP
0

DP 114
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Non Instrumental Factors

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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

DP
0

DP 118
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The Cure

Reload
Or
Gently tap the bottom of the cell with the
loading syringe

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Not long enough between injections

19

18

17
cal/sec

16

15

14
0.00 33.33 66.67 100.00 133.33 166.67
Time (min)

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Not long enough between injections

16

14

12
cal/sec

10

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

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The Cure

Increase the time between the injections

This can be done on the fly by going to the
advanced experimental tab and update the
change to injection parameters>spacing(secs)

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Sticky Proteins or Cleanliness

Change in baseline
cal/sec

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

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Sticky Proteins or Cleanliness

2
cal/sec

-2

-4
0.00 8.33 16.67 25.00 33.33 41.67 50.00

Time (min)

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Sticky Proteins or Cleanliness

16
cal/sec

14

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

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Sticky Proteins or Cleanliness

16

14
cal/sec

12

10
-33.33 0.00 33.33 66.67 100.00 133.33 166.67 200.00 233.33
Time (min)

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Non sigmoidal binding isotherm

No Binding
No Heat
Buffer mismatch
More than one binding event

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Effect of DMSO Mismatch

2% DMSO into 7
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

cal/sec
3

2% DMSO into 2
0% DMSO
1

0
-5 0 5 10 15 20 25 30 35 40 45 50 55

Time (min)

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Buffer Mismatch-No Dialysis

2.5

2.0

1.5
cal/sec

1.0

0.5

without dialysis
0.0
with dialysis

-0.5
0 20 40 60 80 100 120 140 160 180

Time (min)

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The Cure
Dialyze or use desalting column
Check for additives that are in 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

4.52
Control: ligand into buffer
4.50
4.48
4.46
4.44
Heats for experiment
4.42
same as control
cal/sec

4.40
4.38
4.36
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)
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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

19.3

19.2

19.1
cal/sec

19.0

18.9

18.8

18.7

18.6
0.00 33.33 66.67 100.00 133.33 166.67
Time (min)

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Split (oscillating) peaks

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Instrument reference power too low

Oscillating signal:
Power below 0
2

0
cal/sec

-2

-4

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33

Time (min)
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The Cure

Clean and increase reference power in

advanced experimental tab

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 Split Peaks-Sometimes its OK
Time (min)
-10 0 10 20 30 40 50 60 70 80 90 100
4
ITC shows differential
binding of Mn(II) ions to

cal/sec
2
WT T5 5 nuclease
0

-2
2
n = 1.3
Ka = 1.0 x 104 M-1
DH = +1.6 kcal mol-1
kcal/mole
n = 0.85
0
Ka = 3.0 x 105 M-1
DH = -0.59 kcal mol-1

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Molar Ratio 143
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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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Cure-Reverse the Titration
Time (min)
-10 0 10 20 30 40 50 60 70 80 90 100 110 120

29.5M Protein titrated with 0.0

1.1mM Compound -0.5

cal/sec
-1.0

-1.5

11.5M Compound titrated with 2

kcal/mole of injectant
0

179M Protein -2
-4
-6
-8
-10
-12
Time (min)
-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
cal/sec

-0.4
Parameter Ligand in syringe Ligand in cell
-0.6

-0.8
2
n 0.99 0.97
kcal/mole of injectant

0
-2 Kd 104 nM 105 nM
-4
-6 DGo -9.4 kcal/mol -9.4 kcal/mol
-8
-10
DHo -11.9 kcal/mol -12.4 kcal/mol
-12
-14
0.0 0.5 1.0 1.5 2.0 2.5 TDSo -2.5 kcal/mol -3.0 kcal/mol
Molar Ratio 145
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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 correctly folded and active

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ITC General Troubleshooting

Clean cell and syringe thoroughly

Take care with sample preparation
Check initial baseline position
Use appropriate run parameters
Perform water-water titration to rule out
instrumental issues

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ITC Maintenance

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

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ITC Maintenance

Syringe:
20 % Contrad 70 soak for sticky proteins
Store dry
Do not overtighten fill port adaptor
Take care to not bend needle or crack syringe
Change plunger tip when needed

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Thank you!
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