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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
www.nanotemper.de
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
www.nanotemper.de MST Technology
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
► …
www.nanotemper.de MST Technology
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
► …
www.nanotemper.de
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
www.nanotemper.de
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
www.nanotemper.de
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
Some Like it Hot
Some Molecules too
www.nanotemper.de
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
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
www.nanotemper.de
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