Post on 08-Feb-2021
MicroCal iTC200Customer training: Introduction to the technology
Gamze KARAKULLUKÇU, MScApplication Engineergamze@atomikateknik.com | +90 549 745 30 04
mailto:gamze@atomikateknik.com
Isothermal titration calorimetry (ITC)
• What is ITC?• Direct measurement of the heat generated or absorbed when molecules
interact
• ITC determines;• Affinity (KD) in millimolar (mM) to nanomolar (nM) range
• Number of binding sites (N) – stoichiometry
• Binding enthalpy (ΔH) and entropy (ΔS)• Mechanism of binding in a single experiment
Why ITC?
• No molecular weight limitations (ITC)
• Native molecules in solution (biological relevance)
Label-freeBroad dynamic
range Ease-of-use
• Direct measurement of heat change
• No immobilization
necessary
• No/minimal assay development
• Free choice of solvent
Information rich
• Rapid results for KD n, ΔH and ΔS from ITC experiments
0 1 2
-12
-9
-6
-3
0
Xt/Mt
ND
H, k
cal/m
ole
of in
ject
ant
Performing an ITC assay
• Ligand (titrant, injectant etc.) in syringe
• Macromolecule (sample, protein, target etc.) in sample cell
• Heat of interaction is measured
• In a single ITC experiment whole thermodynamic profile of a reaction can be obtained
Reference cell Sample cell
Syringe
How Do ITCs Work?
Reference Calibration Heater
Cell Main HeaterSample Calibration Heater
DP
DT
S RThe DP is a measured power differentialbetween the reference and samplecells to maintain a zero temperaturebetween the cells
DT~0 DP = Differential power∆T = Temperature difference
Ou
ter Shield
Jacket
TD
DP
Seeking TemperatureEquilibration without StirringEquilibration with StirringFirst InjectionTitration
30
0
0
5
DPDT
ShieldJacket
*https://www.youtube.com/watch?v=o_IpWcWKNXI&list=PL2wIBTZfZRjdrXotKLAt1zDEGD3aRBSw4&index=45
Outcome of Basic ITC Experiment
Integration of heats are used to extract affinity (KD), stoichiometry (N) and binding enthalpy (DH) using appropriate
binding model
-4
-2
0
0 0.5 1.0 1.5 2.00.0 0.5 1.0 1.5 2.0
-4
-2
0
Molar Ratio
kca
l/m
ole
of i
nje
ctan
t
DH
N
KD
kcal
mo
l-1o
f in
ject
ant
Molar ratio
µca
l s-1
Time ->
The Energetics
∆H
-T∆S
DG = R T ln KDDG = DH -TDS ∆H, enthalpy is indication of changes in
hydrogen and van der Waals bonding
-T∆S, entropy is indication of changes inhydrophobic interaction andconformational changes
N, stoichiometry indicates the ratio of ligand-to-macromolecule binding
Graphic courtesy of Prof. Dr. Knut Baumann, Technische Universitaet Braunschweig, Germany
Overall binding affinity KD is directly related to ∆G, the total free binding energy
Affinity is just part of the picture
-20
-15
-10
-5
0
5
10
kc
al/m
ole ∆G
∆H
-T∆S
A. Good hydrogen bonding with unfavorable conformational change
B. Binding dominated by hydrophobic interaction
C. Favorable hydrogen and hydrophobic interaction
All three interactions have the same binding energy (∆G)
Favorable
Unfavorable
Binding (Understanding KD)
KD (dissociation constant) has units of concentration
• Describes affinity between protein (P) and ligand (L)
• Equilibrium
• P + L PL
• KD = [P][L]/[PL]
• KD is inverse of affinity constant KA (equilibrium constant)
ITC experiment design (Understanding C value)
• What is the C value? • ([protein in cell]/KD)*N
• Why does it matter?• Data quality = successful experiments
• C value/sigmoidicity is central to obtaining high quality data
• Reliable affinity constant
• Thermodynamic values
• Stoichiometry of an interaction
Four Crucial Steps to Great ITC Data
Sample preparation
The experiment
Data analysis
Experimental optimization
I. Sample Preparation
1. Ensure that macromolecule and titrant solutions are well matched• Matching buffer (Dialysis or Buffer exchange when working with proteins)
• If the ligand is too small for dialysis then dialyze the macromolecule and then dissolve the ligand in the dialysate
• If the ligand is in DMSO solution; dilute the ligand stock solution containing DMSO with the dialysate and then…
• Add a corresponding amount of DMSO to the protein solution
• Matching pH• Choice of buffer
2. Accurately measure [macromolecule] • Protein A280 (Thermo Scientific NanoDrop Spectrophotometer)• Be as accurate as you can weighing the ligand. UV absorption is better if
ligand has a chromophore
Poor sample preparation leads to poor data
• The data shown here shows before and after dialysis
• The large peaks were due to differences in the [NaCl] between buffers
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
DMSO mismatches (example)
• Large heats from DMSO (Dimethyl sulfoxide) dilution, if buffers are not matched
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Time (min)
0.5 cal/sec
pH mismatches
• pH mismatches can arise when using high concentrations of charged ligands i.e. mM concentrations and above. To correct:
Add small drops of NaOH or HCl
Increase the buffer concentration until the ligand charge does not change the pH
Choice of Buffer
• ITC is robust, almost all buffers can be used e.g HEPES, PBS, glycine, acetate
• If reducing agent (proton donor) is required it is best to use either• Tris (2-carboxyethylphosphine) hydrohloride (TCEP) or
• β-mercaptoethanol (BME)
• DTT (dithiothreitol) not recommended because they have small heat of oxidation which occurs as the buffer ages
• Do not use strong acids with ITC!
II. The Experiment
1. Make sure the cell and the syringe is clean (Rinse with 20% Contrador 14% Decon 90 and then water)
2. Key questions:
• How much sample do I need?
• What are the ideal run parameters?
• What controls should I perform?
Dirty Cell leads bad data
0.00 10.00 20.00 30.00 40.00 50.00
8.00
8.50
9.00
9.50
10.00
10.50
11.00
Time (min)
µca
l/se
c
For aggressive cleaning: Rinse with 20%
CONTRAD (14% Decon90) and then water
How much sample is required? (KD & C value)
Do you know the KD?
Estimated KD µM
[Protein] µM
[Ligand] µM
[Protein]/ KD= ‘c’
20
0.5-2 20 200 10-40
2-10 50 500 5-25
10-100 30 40*KD 0.3-3
>100 30 20*KD
Degree of saturation/sigmoidicity (C Value)
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
8.50
9.00
9.50
10.00
10.50
Time (min)
µca
l/se
c
[BCA II], C
5 M, C= 10
10 M, C= 20
50 M, C=100
20 M, C= 40
0.0 0.5 1.0 1.5 2.0
-8.0
-6.0
-4.0
-2.0
0.0
Molar Ratio
kcal m
ol-
1 o
f in
ject
ant
[Furosemide] = 10 *[BCA II]
KD ~ 500 nM
What are the ideal run parameters?
Typical injection parameters:
• Volume: typical 2 l (range 0.1-38 l). An initial injection of 0.4 or 0.5 l is made followed by 18, 2 l injections
• Duration: 4 seconds (0.5 sec/ l)
• Spacing: typical 150-180 seconds
• Filter period: 5 seconds, it’s the time span of data acquisition for data averaging
• Reference power: 3 to 10 cals/sec
• Stir speed: 500-1000 rpm (750 rpm recommended for injection syringes with twisted paddle)
• Feedback: High
What controls should I perform?
• The ligand solution should be injected into the buffer under the same experimental conditions as the titration experiment (Use the same experimental parameters)• Heat of dilution of the ligand
• Heat of injection (machine blank)
• Use this heat change to estimate heat of dilution, should be the same as the heat change at end of ITC titration
III. Data Analysis
- Read Data… (Open multiple runs; Sample and Control experiments together)
- Adjust molar ratio (Concentration check)
- Remove Bad Data Point
- Subtracting the reference
- Fit the model and iterate
- Obtain Final Figure
- Adjust Baseline and Integrations
- Analysing multiple runs and subtracting reference
The raw data & The integrated heats
-3.33 0.00 3.33 6.67 10.00 13.33 16.67 20.00 23.33 26.67 30.00
9.70
9.75
9.80
9.85
9.90
9.95
10.00
10.05
10.10
10.15
Time (min)
µca
l/se
c
-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-32.00
-30.00
-28.00
-26.00
-24.00
-22.00
-20.00
-18.00
-16.00
-14.00
-12.00
-10.00
-8.00
-6.00
-4.00
-2.00
0.00
2.00
Molar Ratio
KC
al/M
ole
of In
ject
ant
Remove first data point
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
-14.00
-12.00
-10.00
-8.00
-6.00
-4.00
-2.00
0.00
2.00
Molar Ratio
KC
al/M
ole
of In
jecta
nt
The first data points are normally
‘bad’ and should be routinely
removed using the ‘Remove Bad
Data Point Button’
Determine mean
Find the mean
Find the mean
The ‘Math’ function
The “one set of sites” model
Math and refit … Imperfect control
Final figure
-3.33 0.00 3.33 6.67 10.00 13.33 16.67 20.00 23.33 26.67 30.00
9.70
9.75
9.80
9.85
9.90
9.95
10.00
10.05
10.10
10.15
Time (min)
µca
l/se
c
Save time?
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
Choice of model
When would you use a more complex model than a one set of sites model?
• Two identical binding sites (same K and ΔH) one set of sites • Two different values of K and/or ΔH two sets of sites
Data that cannot be fit well to simple models may be telling you something important about your system!
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
-16
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of
inje
cta
nt
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
-16
-14
-12
-10
-8
-6
-4
-2
0
Molar Ratio
kcal/m
ole
of
inje
cta
nt
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
-16
-14
-12
-10
-8
-6
-4
-2
0
2
4
Molar Ratio
kcal/m
ole
of in
jecta
nt
ITC shows differential binding of Mn(II) ions to WT T5 5’ nuclease
Two binding sites -2
0
2
4-10 0 10 20 30 40 50 60 70 80 90 100
Time (min)
µcal/sec
-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 Ratiokcal/m
ole
of in
jecta
nt
n = 0.85
KA = 3.0 x 105 M-1
DH = -0.59 kcal mol-1
n = 1.3
KA = 1.0 x 104 M-1
DH = +1.6 kcal mol-1
Example from literature
IV. Experimental Optimization
Bovine carbonic anhydrase II (BCAII) binding to 6 ligands
• Wide affinity range • 5 nM to 10 M
• Wide enthalpy range• -5 to -14 kcals/mol
• All measurements performed in 50 mM HEPES buffer, pH 7.4 and 5% DMSO
Guidelines for high quality data
• Heat of injection• >2.5 cals for the second (first full) peak is ideal
• ~1 cals for second peak is minimum heat
• C value• >1 and 2.5 cals
Guidelines for good enthalpy data
0 2 4 6 8 10 12 14 16 18 20
80
85
90
95
100
105
110
115
120
% 'c
orr
ect
' heat
heat of 2nd
injection
3 l
2 l
1 lheats< 2.5 mcals and C< 13
and
heats < 1 mcals and C2 cals and C >~10 when using 1 or 2 l injections
Guidelines for good KD data
•Conclusion:
• More precise KD with C>5 and
Guidelines for good KD data
-2 0 2 4 6 8 10 12 14 16 18 20
-20
-19
-18
-17
-16
-15
-14
-13
-12
-11
ln K
D
Size of second injection cals)
Ethoxyl
ACZA
AMBSA
CBS
Furosemide
Sulfanilimide
More precise KD when the heats are
> 2 cals or ~ 1 calfor tight binders
Raw data using standard protocol
0.00 10.00 20.00 30.00 40.00
9.40
9.50
9.60
9.70
9.80
9.90
10.00
10.10
10.20
10.30
10.40
10.50
200 M Ethoxylamide into 20 M BCAII
Time (min)
µcal/sec
200 M ACZA into 20 M BCAII
0.00 10.00 20.00 30.00 40.00 50.00
9.80
9.90
10.00
10.10
10.20
10.30
10.40
10.50
10.60
200 M Furosemide into 20 M BCAII
Time (min)
µcal/sec
200 M CBS into 20 M BCAII
0.00 10.00 20.00 30.00 40.00 50.00
9.80
9.90
10.00
10.10
10.20
200 M AMBSA into 20 M BCAII
Time (min)
µca
l/se
c
200 M sulfanilimide into 20 M BCAII
1* 0.5l then 18 * 2 l injections
Ethoxylamide and ACZA data
0.0 0.5 1.0 1.5 2.0
-14.0
-12.0
-10.0
-8.0
-6.0
-4.0
-2.0
0.0
Molar Ratio
kcal m
ol-
1 o
f in
ject
ant
0.0 0.5 1.0 1.5 2.0
-16.0
-14.0
-12.0
-10.0
-8.0
-6.0
-4.0
-2.0
0.0
Molar Ratio
kca
l m
ol-1
of
inje
cta
nt
Ethoxyl-
amide
C ~ 1150
ACZA
C ~ 250
0.00 10.00 20.00 30.00 40.00
9.40
9.50
9.60
9.70
9.80
9.90
10.00
10.10
10.20
10.30
10.40
10.50
200 M Ethoxylamide into 20 M BCAII
Time (min)
µca
l/se
c
200 M ACZA into 20 M BCAII
~ 5 to 6 cals
‘Ethoxylamide’ optimization • Ethoxylamide
• Heat of first full injection was 0.7 cals. This is low, underestimate the DH by ~10 % but rewarded by a good C value
• KD is 6 nM, C=880.Great, at least 2 data points in the transition region
0.0 0.5 1.0 1.5 2.0
-12.0
-10.0
-8.0
-6.0
-4.0
-2.0
0.0
-0.08
-0.06
-0.04
-0.02
0.00
0 10 20 30 40 50 60 70
Time (min)
µca
l/se
c
Molar Ratio
kcal m
ol-
1 o
f in
ject
ant
37 * 1 l injections of
50 M Ethoxylamide into 5 M protein
Reduced concentrations andinjection volume
CBS and furosemide data
0.00 10.00 20.00 30.00 40.00 50.00
9.80
9.90
10.00
10.10
10.20
10.30
10.40
10.50
10.60
200 M Furosemide into 20 M BCAII
Time (min)
µca
l/se
c
200 M CBS into 20 M BCAII
0.0 0.5 1.0 1.5 2.0
-12.0
-10.0
-8.0
-6.0
-4.0
-2.0
0.0
Molar Ratio
kcal m
ol-
1 o
f in
ject
ant
0.0 0.5 1.0 1.5 2.0
-8.0
-6.0
-4.0
-2.0
0.0
Molar Ratio
kcal m
ol-1
of
inje
cta
nt
~ 3 cals
CBS
C ~ 22
Furosemide
C ~ 36
No need for optimization
~ 5 cals
Sulfanilimide and AMBSA data
Sulfanil-
imide
C ~ 2
AMBSA
C ~ 2
0.00 10.00 20.00 30.00 40.00 50.00
9.80
9.90
10.00
10.10
10.20
200 M AMBSA into 20 M BCAII
Time (min)
µcal/sec
200 M sulfanilimide into 20 M BCAII
0.0 0.5 1.0 1.5 2.0
-3.0
-2.0
-1.0
Molar Ratio
kcal m
ol-1
of
inje
cta
nt
0.0 0.5 1.0 1.5 2.0
-8.0
-7.0
-6.0
-5.0
-4.0
-3.0
-2.0
Molar Ratio
kca
l m
ol-1
of
inje
cta
nt
~ 1 cals
~ 2.5 cals
Sulfanilimide optimization
• Sulfanilimide
• Heat is 7.4 cals-good
• KD is 8 M
• C= 6
18 * 2 l injections of 500 M Sulfanilimide into 50 M protein
0.0 0.5 1.0 1.5 2.0
-6.0
-4.0
-2.0
Molar Ratio
kcal m
ol-
1 o
f in
ject
ant
Increased concentrations
AMBSA optimization
• AMBSA
• Heat is 4.8 cals-good
• KD is 10 M
• C= 5
0.0 0.5 1.0 1.5 2.0
-4.0
-2.0
0.0
Molar Ratio
kcal m
ol-
1 o
f in
ject
ant
18 * 2 l injections of 500 M AMBSA into 50 M protein
Increased concentrations
How do I leave my MicroCal ITC after the experiment?1. Rinse cell with detergent solution and water
2. If the system is to be left for any period of time, the cells should be filled with buffer or water
3. The injection syringe should be stored dry
4. If the system is not going to be used on regular basis, the cells should be filled with 0.01% aqueous sodium azide to prevent bacterial growth
5. Shut down the instrument when not in use
Routine user maintenanceDaily/After every run
1. Centrifuge or filter all solutions before use to avoid particles and precipitates in the cell or syringe
2. Rinse the cell with 20% Contrad 70 or 14% Decon 90, followed by deionized water
3. When needed, heat cell to 60 C and fill cell with 20% Contrad 70 or 14% Decon 90, soak for 1 hour, cool cell and rinse with deionized water
4. When needed, clean syringe with detergent solution, and cleaning wire
Routine user maintenanceWeekly maintenance
1. Refill the methanol, buffer and water bottles of ITC200 wash module
2. Rinse reference cell and refill with deionized water
Routine user maintenanceWhenever necessary
1. Replace Teflon plunger tip (in every 200-300 runs is recommended)
2. Replace damaged syringe
Tips and Tricks
• Water-water test results is a good way to validate the instrument
• Don’t use strong acids
• Don’t leave Contrad 70/Decon 90 in the cell too long (ex. Overnight) you may destroy the cell; increase the temperature to 60C for cleaning better
• Don’t go below 0C because the water freezes and breaks the cell
• 200-300 injections is the life time of the plunger tip
• Lab conditions are important
• Don’t place MicroCal directly near and below Sun light – window isle – windy (air conditioner) affects the DP (differential power)
• Bent syringe, height of the syringe cause metal rubbing and create heat (decreases the reference power)
• Stir speed creates heat and decreases DP
• Bubbles create heat capacity difference
Thank you!