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!