Some like it hot: Biomolecule Analytics using Microscale Thermophoresis

NanoTemper Technologies GmbH Flößergasse 4, 81369 München, Germany

www.nanotemper-technologies.com

www.nanotemper.de

www.nanotemper.de MST Technology

Microscale Thermophoresis (MST)

Electrophoresis: Electric Field

Charge, (Size)

Thermophoresis: Temperature Gradient

Charge, Size and Hydration Shell

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Theory of Thermophoresis

size charge 2

chot/ccold= exp (-ST ΔT)

+

-

hydration shell

Duhr and Braun PNAS 103, 19678–19682 (2006) Duhr and Braun PRL 96, 168301 (2006)

MST Technology

www.nanotemper.de

Optical Microscale Thermophoresis

Basic Setup of the Instrument

LED excitation & fluorescence detection

IR laser dichroic mirror

objective

capillary

23°C

20°C

1K/10 µm corresponds to 1000K/cm

IR laser induced temperature field (BCECF-dye in TRIS-buffer)

► immobilization-free in any buffer

► 4 µl of sample per titration point

► pM - nM concentrations of fluorescent molecule

► 40 sec measurement per titration point

MST Technology

www.nanotemper.de MST Technology

MST Binding Curve

Time [s]

Saturation

Baseline

Baaske et al., Angewandte Chemie, 2010

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Information accessible with MST

► binding affinity (Kd, EC50,..)

► stoichiometry

► binding energetics (dG, dH, dS)

► binding mode of action (agonist, antagonist)

► stability (thermal stability Tm, pH/buffer conditions)

► dimerization/polymerization, aggregation

► …

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Interactions accessible with MST

► antigen - antibody

► protein - protein

► protein - DNA/RNA

► nucleic acid - nucleic acid

► protein - small molecule, peptides, ions

► protein or small molecule - HMW complexes (e.g. ribosome)

► protein or peptide - liposome/vesicle

► ligand binding to membrane receptors

► …

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Interaction of fluorescently labeled calmodulin with Ca2+ - ions

Wienken et al, Nature Communications, 2010

Protein – Ion Interaction

Ca2+

Conformational change of Calmodulin upon Ca2+-binding

► Interactions that do not alter the MW of a protein, only charge and the conformation (hydration shell)

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Cooperative binding of Synaptotagmin to Ca2+ and the phospholipid PIP2

Protein – Ion Interaction

► Multicomponent interactions and cooperativity are easily detectable by MST

Synaptotagmin Wildtype PIP2 and Ca2+ binding

Synaptotagmin Mutant No PIP2 binding

Boogart et al, JBC, 2010

www.nanotemper.de Interaction to high molecular weight complexes

Binding of a labeled multimeric ribosomal interactor to the ribosome

► Broad sensitivity range of MST: from ions to HMW complexes

“multimeric ribosomal interactor”

Ribosome

Results were kindly provided by Julian Deeng, Genzentrum LMU München, Germany

www.nanotemper.de LabelFree Receptor – Ligand Interaction

Neurotensin Receptor (GPCR) interactions with different molecules

► Allows fast and label-free binding experiments with sensitive GPCRs

Prof. Anthony Watts, University of Oxford, Biochemistry, UK Seidel et al, Methods, 2012

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MST Benefits

MST puts your molecules into the focus

► low sample consumption

► immobilization free

► fast measurement: obtain a Kd in 10 min

► rapid assay optimization

► access to affinities, stoichiometries, binding energetics

► low consumable costs

► maintenance-free instrument

► straightforward handling

Key Features

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1856: Carl Ludwig

1879: Charles Soret

2008

2010: Monolith NT.115

2012: Monolith

NT.LabelFree

Stefan Duhr Philipp Baaske

June 2013: >70 Publications

Fast thermo-optical particle characterisation PCT Patent 2008/061706

June 2013: >1000 Users 4 Continents 2013:

Monolith NT.115 Pico

www.nanotemper.de Monolith NT Series

Instruments for Microscale Thermophoresis

Monolith NT.115 Monolith NT.115Pico Monolith NT.LabelFree

► broad application range

from ions to ribosomes

► buffer independency

even serum or cell lysate

► purification free

fluorescent fusion proteins

► dynamic range

1 nM to mM

► high sensitivity

limit of detection at 50 pM

► low sample consumption

pM concentrations

► better resolution

for high affinity interactions

► dynamic range

10 pM to mM

► TRULY label-free

intrinsic fluorescence

► buffer independency

except complex bioliquids

► dynamic range

10 nM to mM

Thank You For Your Attention!

Please feel free to contact us:

NanoTemper Technologies GmbH Flößergasse 4, 81369 München, Germany

www.nanotemper-technologies.com

philipp.baaske@nanotemper.de

Some Like it Hot

Some Molecules too

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MST in Comparison

Criterion Surface Plasmon Resonance (SPR) Isothermal Titration Calorimetry (ITC) Microscale Thermophoresis (MST)

Sample consumption ●●● ●●●●● ●

Cost of consumables high low low

Capacity/run 1 sample 1 sample 1 - 16 samples

Time required for experiment plus analysis

hours hours minutes

Sample range (Dalton) 102 - 106 101 - 106 101 - 107

Type of information Kd, kinetics, stoichiometry Kd, enthalpy, stoichiometry Kd, stoichiometry, aggregation, enthalpy (van’t Hoff plot), enzyme kinetics, oligomerization

Dynamic range of Kd 10-9-10-4 M 10-5-10-3 M 10-11-10-2 M

Measure in lysate or whole cell extract no no yes

Purity required highly purified highly purified purification free

Method handling difficult intermediate easy

Assay optimization time week(s) day(s) hour(s)

Service cost high low none

Key Features

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Dimerization of Grb2

Protein – Protein Interaction

► Detection of dimerization at low protein concentrations

Lin and Ladbury, CELL, 2012

Grb2

EGFR Peptide

Microscale Thermophoresis

Light scattering

www.nanotemper.de LabelFree Protein – Small Molecule Interaction

p38 interactions with different compounds

Seidel et al, Angwandte Chemie, 2012

► Allows fast and label-free screening of various compounds binding to different types of proteins

www.nanotemper.de Biophysical Comparison

Ligand Binding to Carbonic Anhydrase – Kd [µM]

Ligand Biacore (Literature)

Proteon (Literature)

Biacore (Sanofi)

MST NT.115 NT.LabelFree

Benzenesulfonamide 0.8 ± 0.3 1.1 ± 0.1 0.41 0.80 ± 0.10 1.09 ± 0.09

Sulfanilamide 3.1 ± 1.1 4.2 ± 0.9 2.2 3.40 ± 0.2 n.d.

Furosemide 1.0 ± 0.2 0.8 ± 0.1 0.42 1.00 ± 0.2 0.89 ± 0.04

4-Sulfamoybenzoic acid 0.97 ± 0.17 1.6 ± 0.4 0.41 1.3 ± 0.4 0.4 ± 0.04

Data kindly provided by Dr. Alexey Rak, Sanofi-Aventis, Paris

“… We use Microscale Thermophoresis (MST) in addition to other methods including ITC and SPR and find a good agreement between the methods … “

“… There is very good agreement with other technologies such as Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC), … “

Dr. Alexey Rak Head of Structural Biology & Biophysics - BBC, SDI Paris/LGCR, Sanofi R&D, France.

Prof. John E. Ladbury Department of Biochemistry and Molecular Biology The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

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MST in Comparison

Criterion Surface Plasmon Resonance (SPR) Isothermal Titration Calorimetry (ITC) Microscale Thermophoresis (MST)

Sample consumption ●●● ●●●●● ●

Cost of consumables high low low

Capacity/run 1 sample 1 sample 1 - 16 samples

Time required for experiment plus analysis

hours hours minutes

Sample range (Dalton) 102 - 106 101 - 106 101 - 107

Type of information Kd, kinetics, stoichiometry Kd, enthalpy, stoichiometry Kd, stoichiometry, aggregation, enthalpy (van’t Hoff plot), enzyme kinetics, oligomerization

Dynamic range of Kd 10-9-10-4 M 10-5-10-3 M 10-11-10-2 M

Measure in lysate or whole cell extract no no yes

Purity required highly purified highly purified purification free

Method handling difficult intermediate easy

Assay optimization time week(s) day(s) hour(s)

Service cost high low none

Key Features

www.nanotemper.de

MST re-shuffled NMR results

MST – Biophysical Comparison

► MST re-shuffles results for Top-10 NMR and in vitro hits, but nicely correlates with crystallographic data

NMR/in vitro score

9 4 8 7 2 10 1 3 5 6

MST score

1 2 3 4 5 6 - - - -

Pim1 Kd (µM) 8 44 83 150 950 800 13 0 0 0

X-ray 1 1 1 1 1 0 1 0 0 0

MW 177 221 240 176 195 210 212 236 152 165