Isothermal titration calorimetry:

Principles and experimental design

Stoyan Milev, Ph.D

GE Healthcare

stoyan.milev@ge.com
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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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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?
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/K
D
5
0
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MicroCal VP-ITC MicroCal iTC
200
MicroCal Auto-iTC
200

1400 L cell
Manual sample
loading
Up to 5
samples/day

Sensitive
Fast
Easy to use
K
D
from mM to nM
200 L cell
Upgradable to full
automation
Unattended operation
Up to 75 samples/day
(using single injection
method)
K
D
from mM to nM
Sample cell is 200 L
Easy to use
96-well plate format








MicroCal

ITC

systems
Why ITC?
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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
K
D
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
K
B
binding constant

K
D
= 1/

K
B
=

DG = RT lnK
D

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
-TS
Overall binding affinity K
D
correlates with IC
50
or EC
50
.
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
-TS, 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
k
c
a
l
/
m
o
l
e
G
H
-TS
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
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t

0.05
0.5
5
50
500



C

=
[M]
K
D

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)
time (min)

c
a
l
/
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)
k
c
a
l
/
m
o
l

o
f

i
n
j
e
c
t
e
d

R
a
s
c = 5
c = 40
Kd = 1.2 M
C Values
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C Values in ITC



C = {[M]
tot
/ K
D
}

* 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
k
c
a
l

m
o
l
-
1

o
f

i
n
j
e
c
t
a
n
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
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
10< [Protein]/K
D
<100
Fitted: N, K
D
, DH
0.0 0.5 1.0 1.5 2.0
-4
-2
0


Molar Ratio
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
-4
-2
0
0 0.5 1.0 1.5 2.0
[Protein]/K
D
>> 1000
Fitted: N, DH
0.0 0.5 1.0 1.5 2.0
-4
-2
0


Molar Ratio
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
-0.4
-0.2
0
0 4 8 12 16
[Protein]/K
D
< 1
N fixed
Fitted: K
D
, DH
High C
Low C
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ITC experimental design
K
D
(Biacore) M [Protein] M [Compound] M [Protein] / K
D
<0.5 10 100 >20
0.5-2 30 300 15-60
2-10 50 500 5-25
10-100 30 40*K
D
(Biacore) 0.3-3
>100 30 20*K
D
(Biacore) <0.3
Fixed
stoichiometry
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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
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
0 2
-1.7
-1.6
-1.5
-1.4
-1.3
-1.2
-1.1
Molar Ratio
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
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
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
0--------------->26
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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 10
10
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)

c
a
l
/
s
e
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
k
c
a
l
/
m
o
l
e

o
f

i
n
j
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c
t
a
n
t
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MicroCal iTC
200
:
Minimum Heat=Minimum [Protein]
For iTC
200
:
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
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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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iTC
200
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 K
D
s of 1 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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Choice of buffer
Buffers have ionization enthalpies:

BH B
-
+ H
+

Use buffers with DH
ion
~ 0
Including; phosphate, acetate, formate, citrate,
sulfate, cacodylate, glycine
Quaternary amines (e.g. Tris) have high DH
ion


DH
ion
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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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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)

c
a
l
/
s
e
c
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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
200
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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iTC
200

Macromolecule (in the cell):
Need 300 l

Ligand (in the syringe):
Need 70 l

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ITC Enthalpy Changes
DH
observed
by ITC is total of :
DH
binding
DH
ionization
DH
conformation
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)

c
a
l
/
s
e
c
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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)

c
a
l
/
s
e
c
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
Troubleshooting
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Why Do ITC Experiments Not Work
as Expected?

Concentration errors because K
D
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
0
5
D
P

DP
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Syringe Height
0
5
DP
iTC
200
- Syringe Holding Nut Loose
D
P

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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
0
5
D
P

DP
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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
0.00 33.33 66.67 100.00 133.33 166.67
14
15
16
17
18
19
Time (min)

c
a
l
/
s
e
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)

c
a
l
/
s
e
c
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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
-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)

c
a
l
/
s
e
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)

c
a
l
/
s
e
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)

c
a
l
/
s
e
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
10
12
14
16
Time (min)

c
a
l
/
s
e
c
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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
-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)

c
a
l
/
s
e
c
2% DMSO into
2% DMSO-same stock
2% DMSO into
2% DMSO-separate stocks
2% DMSO into
0% DMSO
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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)

c
a
l
/
s
e
c
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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
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)

c
a
l
/
s
e
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)

c
a
l
/
s
e
c
140
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Split (oscillating) peaks
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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)

c
a
l
/
s
e
c
Oscillating signal:
Power below 0
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The Cure
Clean and increase reference power in
advanced experimental tab
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Split Peaks-Sometimes its OK

ITC shows differential
binding of Mn(II) ions to
WT T5 5 nuclease
-2
0
2
4
-10 0 10 20 30 40 50 60 70 80 90 100
Time (min)

c
a
l
/
s
e
c

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
0
2
Molar Ratio

k
c
a
l
/
m
o
l
e



n = 0.85
K
a
= 3.0 x 10
5
M
-1
DH = -0.59 kcal mol
-1
n = 1.3
K
a
= 1.0 x 10
4
M
-1
DH = +1.6 kcal mol
-1
Feng, et al, Nat. Struct. Mol. Biol. 11, 450-456 (2004)
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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)

c
a
l
/
s
e
c
0.0 0.5 1.0 1.5 2.0 2.5
-12
-10
-8
-6
-4
-2
0
2
Molar Ratio
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
-0.8
-0.6
-0.4
-0.2
0.0
-10 0 10 20 30 40 50 60 70 80 90 100
Time (min)

c
a
l
/
s
e
c
0.0 0.5 1.0 1.5 2.0 2.5
-14
-12
-10
-8
-6
-4
-2
0
2
Molar Ratio
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
Parameter

Ligand in syringe

Ligand in cell

n

0.99

0.97

K
d


104 nM

105 nM

DG
o


-9.4 kcal/mol

-9.4 kcal/mol

DH
o


-11.9 kcal/mol

-12.4 kcal/mol

TDS
o


-2.5 kcal/mol

-3.0 kcal/mol

29.5M Protein titrated with
1.1mM Compound
11.5M Compound titrated with
179M 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 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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