Microscale Thermophoresis

Microscale Thermophoresis
Technology and Applications
Contents
1. Technology
2. Technology Platform
3. Handling
4. Applications
5. Publications
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1. Technology
Microscale Thermophoresis
Microscale Thermophoresis (MST) is a powerful new
technology, and easy to handle. It detects changes in
the hydration shell of molecules and measures
biomolecule interactions under close-to-native
conditions: immobilization-free and in bioliquids of
choice.
Infrared-lasers are used to achieve precise microscale
temperature gradients within thin glass capillaries that
are filled with a solution of choice (buffer or serum, cell
lysate and other bioliquids). Molecules move along these
temperature gradients. Extensive research conducted at
the Biophysics Department of the Ludwig-MaximiliansUniversity Munich (LMU) identified the solvation entropy
and the hydration shell of molecules as the driving force.
Any change of the hydration shell of biomolecules due to
changes in their primary, secondary, tertiary and/or
quaternary structure affects the thermophoretic
movement and is used to determine binding affinities
with high accuracy and sensitivity.
NanoTemper´s unique technology is ideal for basic
research applications requiring flexibility in the
experimental scale, as well as for pharmaceutical
research applications including small molecules profiling,
which are difficult to access with established
technologies as they need a high sensitivity.
The experimental procedure is straightforward and
eliminates expensive and tedious sample preparation. In
combination with the capillary format it reduces the
overall costs and the setup costs which are typically
associated with standard molecular interaction
technologies.
The technology uses fluorescence in combined with IRLaser optics for local heating of the sample. The heating
laser is focused through the same objective used for
fluorescence detection. This allows a precise local
microscopic heating of the sample within the capillary
and simultaneously and observation of local changes of
fluorescence intensity due to the motion of labeled
molecules in the glass capillaries.
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Fluorescently labeled molecules or particles are initially
distributed evenly and diffuse freely in solution. By
switching on the IR-Laser, the molecules experience a
thermophoretic force in the temperature gradient and
typically move out of the heated spot. In the steady state,
this molecule flow is counterbalanced by ordinary mass
diffusion. After turning off the laser, the particles diffuse
back to obtain a homogeneous distribution again. The
following stages are recorded for each sample:
fluorescence signal before turning the IR laser on, fast
temperature-dependent changes in fluorescence intensity,
thermophoresis and back diffusion after switching the laser
off.
2. Technology Platform
NanoTemper´s Monolith platform provides instruments,
consumables and reagents for analyzing biomolecule
interactions with fluorescence and label-free. It utilizes
Microscale Thermophoresis to enable real-time,
immobilization free analysis of biomolecules providing
information on the affinity, stoichiometry and aggregation
properties of biomolecules in buffers and complex
biological liquids including blood serum and cell lysate.
Monolith Series Instruments
The Monolith NT.115 and NT.LabelFree are
NanoTemper´s instruments for basic research and
pharmaceutical applications. The NT.115 uses
fluorescence dyes to read out the thermophoretic effect,
while the NT.LabelFree uses intrinsic tryptophane
fluorescence. Both are based on NanoTemper’s
Microscale Thermophoresis technology. Equipped with the
standard sample tray, each instrument can process
automatically up to 16 samples per run in 10 minutes.
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Key Benefits
Unmatched Sensitivity
Unique performance
Microscale Thermophoresis can monitor the binding of
single ions (40Da) or small molecules (300Da) to a target
as well as the binding of ribosomes (2.5MDa).
Microscale Thermophoresis is easy to handle and allows to
measure the binding of biomolecules as well as the activity
of enzymes. It is ideal for basic research applications
requiring flexibility in the experimental scale, as well as for
pharmaceutical research applications, including small
molecules profiling, which are difficult to access with
established technologies as they require a high sensitivity.
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Close-to-native conditions
measure affinities (KD, dissociation constant)
between any (bio)molecules directly in bioliquids
study membrane bound proteins directly in
liposomes or in detergent solutions
study multi component reactions, complex
formation, order of assembly and interfering
factors
study the effect of serum, cell lysate or other
bioliquids on biomolecules
separate aggregation and other artifacts from true
binding events
measure with fluorescence label and label-free
access larger screening projects in a label-free
manner using fluorescently labeled tool
compounds
discriminate between different binding sites on a
target of interest
study the stoichiometry and determine the number
of binding sites of biomolecules
study the binding energetics dG (free energy ), dH
(enthalpy) and dS (entropy)
study the inhibitor affinity, Ki either directly or in a
competition experiment
Microscale Thermophoresis monitors binding and
biochemical activity of biomolecules under close-to-native
conditions:
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immobilization-free
label-free
In a solution of choice, ranging from standard and
proprietary buffers to complex bioliquids including
blood serum or cell lysates
at a temperature of choice
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Capillary format
Requires little sample material
Dedicated Data Acquisition
and Analysis Software
The capillary format is inexpensive, easy to handle and
offers maximum flexibility in the experiment scale. The
sample tray format allows to process automatically up to
16 capillaries (e.g. for a detailed KD-analysis), or
alternatively to perform smaller pilot experiments or end
point measurements involving 2-3 capillaries only.
Microscale Thermophoresis requires approximately
100fold less sample material compared to standard
technologies. Capillary volume: 3-5 µl at concentrations as
low as 1 nM of the labeled molecule.
The Monolith NT.115 instrument is supported by a
software for data acquisition and analysis.
Monolith Models
Monolith NT.115
Monolith
NT.115
NT.115
Blue/Green
NT.115
Blue/Red
NT.115
Green/Red
LED 1
/nm
Ex:470
LED 2
/nm
Ex:550
Em:520
Em:600
Ex:470
Ex:625
Em:520
Em:680
Ex:520
Ex:625
Em:570
Em:680
3 models of the Monolith NT.115 instrument are offered,
which differ in the excitation/detection spectrum to detect
blue, green and/or red fluorescent dyes, as defined by the
respective color of the excitation light.
Blue Dyes
Green Dyes
Red Dyes
FITC/FAM/GFP/YFP
Cy3/RFP/mCherry
no detection
FITC/FAM/GFP/YFP
no detection
Cy5/Alexa647
YFP
Cy3/RFP
Cy5/Alexa647
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Monolith NT.LabelFree
Monolith
NT.LabelFree
NT.Label
Free
LED 1
/nm
Ex:280
Em:360
The Monolith NT.LabelFree instrument has an excitation
wavelength of about 280nm and an emission wavelength
around 360nm. It allows to use any molecule that has a
fluorescence in this range to be used as the labeled
constituent of a MST experiment (e.g. tryptophane, 2Aminopurin, 8-vinyl-deoxyadenosine, etc)
Molecules (examples)
Proteins containing Tryptophane
2-Aminopurin
8-vinyl-deoxyadenosine
BIRB-796
Monolith Consumables
NanoTemper offers kits and capillaries for use with
Microscale Thermophoresis that enable you to get high
quality MST results. The products are specially designed
for the MST instruments NT.115 and NT.LabelFree.
Capillaries
NanoTemper provides you with capillaries that fullfill the
high requirments of MST in terms of reproducibility,
glass and surface quality as well as background
fluorescence. The capillaries come with different surface
coatings to stabilize even the most complex samples in
solution.
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Labeling Kits
Our labeling kits contain fluorescent dyes that are widely
tested with MST. The labeling protocol ensures good
labeling efficiency and purification. NanoTemper offers
dyes that are optimized for protein compatibility and
MST Temperature Jump. The fluorescence emission
and detection fits perfect to the BLUE, GREEN and RED
channel in the NT.115 instruments.
Assay Development and
Control Kits
Our Assay Development and Control Kits allow you to
get started with MST quickly and train new lab members.
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3. Handling
Microscale Thermophoresis is easy to handle and
involves the following steps:
Labeling (only NT.115)
One of the binding partner is labeled using standard
fluorescent labeling protocols. Blue, green or red dyes
and all coupling chemistries are compatible with our
technology. NanoTemper provides dyes optimized for
protein compatibility and MST analysis. In case the
Monolith NT.LabelFree is used, no labeling is necessary.
Mixing the Reaction
A titration series of up to 15 dilutions is prepared, where
the concentration of the fluorescent binding partner is
kept constant and the concentration of the unlabeled (i.e.
non fluorescent) molecule is varied.
Transfer in Capillary
After an incubation time sufficient for the reaction to
reach equilibrium (e.g. 5 min.), the reaction is transferred
into a glass capillary. The capillary is placed on the
sample tray. The tray, which can accommodate up to 16
capillaries, is placed in the instrument.
Measurement and Data Analysis
Simply start the software and the instrument
automatically recognizes the presence of properly filled
capillaries on the tray. Within 10 minutes it automatically
measures the thermophoresis signal of each sample and
calculates the dissociation coefficient. The analysis
software allows to highlights protein aggregation events,
false positives and discriminates epitope/binding sites.
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4.Applications
Interaction Direct
This category refers to most frequently used MST
applications. The fluorescent binding partner is kept at
constant concentration, while the concentration of the
binding partner is increased. The binding signal is
generated directly by the change in thermophoretic
mobility of the fluorescent molecule.
Protein-Protein Interaction
GFP binding to GFP-binding
Protein
Protein Kinase A interaction with PKI
The binding behaviour of proteins can be easily
measured with MST. In this experiment, MST was used
to study the binding affinity of mutant GFP Binding
Protein (GBP) to GFP. Wildtype GBP showed a high
affinitiy of 2.3 ± 2.1 nM, The exchange of an arginine at
the binding interface (GBP mutant R37A) reduced the
affinity to 80 ± 38 nM. RBP was used as a negative
control and showed no binding to GFP.
Conformational control of protein kinases is an important
way of modulating catalytic activity. Crystal structures of
the C (catalytic) subunit of PKA (protein kinase A) in
complex with physiological inhibitors and/or nucleotides
suggest a highly dynamic switching between open and
more closed conformations. Here we show the detailed
binding analysis of the physiological PKA inhibitor PKI
(heat-stable protein kinase inhibitor), in the presence
and absence of nucleotide cofactors. It could be shown,
that the affinity of the inhibitor PKI is strongly enhanced
in the presence of ATP and Magnesium ions. For this
experiment, the inhibitory protein PKI has been labeled
fluorescently with the dye NT-647 (NanoTemper
Technologies) and the C subunit of PKA is titrated in
presence of ATP/Mg2+ and absence of ATP/Mg2+. A
concentration of 20nM of fluorescently labeled PKI is
mixed with a serial dilution of the catalytic subunit of
PKA (C-subunit). In presence of Magnesium and ATP a
high affinity of 2nM is obtained (left). In absence of ATP
and MgCl2, a strong reduction in the affinity is observed
(KD = 500nM, right).
The catalytic subunit of PKA and the heat stable inhibitor
PKI were kind gifts from F.W. Herberg (University of
Kassel, Germany) and B. Zimmermann (Biaffin GmbH &
Co KG, Germany)
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Small Molecules
17-DMAG binding to Hsp90 Protein
Stephen H. McLaughlin
MRC Laboratory for Molecular Biology, Cambridge, UK
For proper folding, many proteins involved in signaltransduction pathways, cell-cycle regulation and
apoptosis depend upon the ATP-dependent molecular
chaperone Hsp90. Consequently Hsp90 turned out to be
an attractive target for cancer therapeutics. In this study
we demonstrate the binding of the geldanamycin
derivative 17-DMAG to Hsp90 using Microscale
Thermophoresis (MST). The study also highlights the
high content information of the MST measurements as
one important benefit of Microscale Thermophoresis.
The cytosolic heat shock protein 90 (Hsp90) is the focus
of several drug discovery programs for anti-cancer
therapy. The action of Hsp90 underpins the maintenance
of the transformed state through its function in the
conformational maturation and activation of many client
proteins involved in many of the pathways that hallmark
cancer. Consequently, cancer cells are vulnerable to
Hsp90 inactivation (Whitesell et al., 2005).The
interaction of HSP 90 with 17-DMAG was measured with
fluorescent labeling the HSP90 (top) as well as label free
(bottom), using the intrinsic tryptophane fluorescence of
HSP90.
Download the complete application note for further
details.
Binding of Small Molecules to labeled p38 p38 is a serine/threonine protein kinase in the mitogenactivated protein kinase (MAPK) family. p38α is
considered as the key isoform involved in modulating
inflammatory response in rheumatoid arthritis and
inflammatory pain. Two well characterized small
molecule antagonists SB 203580 and the clinical
candidate BIRB-796 were used in this study. Whereas
the first compound competes with ATP for the binding
site on the kinase, BIRB-796 binds adjacent to the active
site and directly inhibits enzymatic activity by affecting
the conformation of the ATP site. The binding of the low
molecular weight compound to the proteins is readily
observed as a change in the thermophoretic property of
the fluorescently labeled protein. The dissociation
constant is determined to 6±2 nM in good agreement
with literature values. This experiment shows that
thermophoresis is sufficiently sensitive to observe
interactions that do not considerably alter the size or
mass of a protein.
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Agonist binding to GluR2 ion channel
AMPA receptors (GluR1–4) are a subtype of the
ionotropic glutamate receptor family of ligand-gated ion
channels and have a high affinity for the full agonist
AMPA. AMPA receptors also bind and activate in
response to the nonselective, full agonists L-glutamate.
Crystallographic studies reveal that full and partial
agonists bind to the cleft of the ‘‘clamshell-shaped’’
GluR2 S1S2J ligand-binding core: The full agonists
AMPA, glutamate brings the domains of the ligandbinding core 21° closer together, relative to the apo
state. The affinity of L-Glutamate to fluorescently labeled
GluR2 was measured by MST. The affinity is in very
good agreement with literature values ((Armstrong et al,
PNAS 100, 10 (2003)))
Material was kindly provided by Prof. Dirk Trauner,
Chemical Genetics and Chemical Biology, LMU Munich
Binding of Triostin Analogues to DNA Eike-F. Sachs and Ulf Diederichsen
Universität Göttingen, Institut für Organische und
Biomolekulare Chemie, Tammannstrasse 2, D-37077
Göttingen, Germany
In this work, we show that Microscale Thermophoresis
(MST) is capable of measuring small molecule binding to
fluorescently labeled DNA molecules. The binding
affinity of derivatives of the antibiotic Triostin to a DNA
molecule is shown. This set the stage for application of
Microscale Thermophoresis as a tool for screening for
sequence specific drugs that can function as an
antibiotic or anti-cancer agent. Triostin A is the most
important member of the family of quinoxaline
antibiotics. Its excellent cytostatic properties originate
from bisintercalative binding to double-stranded DNA via
the minor groove, spanning two base pairs (Waring and
Makoff 1974, Addess and Feigon 1994). This work
shows that MST is a method of choice for the analysis of
small molecule binding to DNA molecules. It allows fast
and precise determination of affinities and check for
dependencies on compound structure and DNA
sequence.
Download the complete application note for further
details.
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Protein Nucleic Acid
HMG protein binding to dsDNA
Gernot Längst
Biochemistry, University of Regensburg, Germany
AT-hooks are short peptide motifs that bind to the minor
groove of AT-rich DNA sequences. The binding of the
AT-hooks to DNA results in changing the regular B-form
structure of DNA. The core motif of a canonical AT-hook
is a GRP tripeptide flanked by basic amino acid patches.
The motif is highly conserved from bacteria to mammals
and crucial for the DNA binding properties of a wide
variety of proteins, ranging from transcription factors to
chromatin remodelers. The well-characterized HMGA
class of proteins, belonging to the 'High Mobility Group'
(HMG) family, solely contains AT-hooks as DNA binding
domains. HMG proteins are involved in many DNA
dependent biological processes, involving transcription,
replication and repair. In this experiment, the
concentration of the fluorescently labeled DNA is kept
constant and the target is titrated. The MST data for
different targets (GST-AT1+2, GST-AT1) that contain 1
or 2 AT-hooks are plotted against the titrated target
concentrations. GST-AT1 and GST-AT1+2 show a
sigmoidal binding curve. The measured values are fitted
with the Hill-equation. The plot indicates that GSTAT1+2 has a five times higher affinity (EC50=4 µM) to
the DNA than GST-AT1 (EC50=20 µM).
Aptamer Interaction with Thrombin
The affinity of a DNA-aptamer to the protein thrombin is
measured in different buffers and 50% human blood
serum. The affinity is highest (KD = 32 nM) in the
"selection buffer". In SSC buffer the affinity is reduced to
about 200 nM. The lowest affinity is observed in 50% of
human serum (KD: 900 nM). With appropriate control
experiments, these effects can be attributed to certain
ions, proteins, the pH or viscosity of the solution
(Angewandte Chemie, 2010, DOI:
10.1002/anie.200903998).
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Protein Ku70/Ku90 interaction with dsDNA
The DNA repair protein Ku acts as a heterodimer of
Ku70 and Ku80 and binds to DNA ends produced during
the generation of programmed double-strand breaks
induced by V(D)J or class switch recombination, or
accidently by variety of DNA damaging agents. It has
been shown that Ku binds much stronger to DNA ends
than to internal DNA regions. Although, there are a
number of reports indicating the possible binding of KU
to nicked DNA, or to single-to-double-stranded DNA
transition, undisputable evidences exist that KU
preferentially binds to DNA ends. The DNA end binding
activity of KU highlights its major functions in genome
stability and maintenance and in the survival of cells
after introduction of DSBs. Here we have measured the
binding of Ku to fluorescently labeled 50 bp dsDNA
using the MST-technology. In the binding reaction,
AlexaFluor 532-labeled- dsDNA was incubated with the
indicated amount of unlabeled Ku. As expected, we
observed strong binding of Ku to DNA with a calculated
KD of about 2 nM, which correlates well with previously
reported SPR and EMSA data.
Material was kindly provided by Prof. Iliakis,
Universitätsklinikum Essen, Germany
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Protein Peptide
Binding of Histone peptides to Chromatin assembly
factor I (CAF-I) p48 subunit
Wei Zhang
University of Cambridge, Department of Biochemistry,
Cambridge, UK
p48, the small subunit of chromatin assembly factor 1
(CAF-1), is a member of a highly conserved subfamily of
WD-repeat proteins. There are at least two members of
this subfamily in human (p46 and p48). p48 copurifies
with a chromatin assembly complex (CAC), which
contains the three subunits of CAF-1 (p150, p60, p48)
and the Histones H3 and H4, and promotes DNA
replication-dependent chromatin assembly. In this study
we analyze the binding of H3 and H4 peptides to p48
using Microscale Thermophoresis (MST). Five major
classes of histones exist: H1/H5, H2A, H2B, H3, and H4.
Histones H2A, H2B, H3 and H4 are known as the core
histones, while histones H1 and H5 are known as the
linker histones. Two of each of the core histones
assemble to form one octameric nucleosome core
particle, and 147 base pairs of DNA wrap around this
core particle 1.65 times in a left-handed super-helical
turn (see Fig.1). In contrast the linker histone H1 binds
the nucleosome at the entry and exit sites of the DNA,
thus locking the DNA into place and allowing the
formation of higher order structure. Histone H5 performs
the same function as histone H1, and replaces H1 in
certain cells. Histone proteins also play essential
structural and functional roles in the transition between
active and inactive chromatin states. Chromatin
Assembly Factor-1 (CAF-1) assembles newly
synthesized histones H3/H4 into DNA in the first step of
nucleosome assembly. Accordingly in human cells, CAF1 is complexed to newly synthesized and acetylated
histones H3 and H4. Human CAF-1 consists of three
subunits: p150, p60 and p48. The small CAF-1 subunit
p48 is a member of a highly conserved subfamily of WDrepeat proteins. Here we checked the interaction of p48
with Histone H3 derived peptides. Fig. 2 shows the
resulting binding curve for the H3NS peptide - NT647labeled p48 interaction with a calculated Kd of 15.76 ±
2.18µM.
Download the complete application note for further
details.
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Nucleic Acids
DNA Triple-Helix Formation
Michael Filarsky,
Lab of Prof. Gernot Längst, University of Regensburg
In this experiment a fluorescently labeled double
stranded DNA (dsDNA) was used, where as one of the
two strands was labeled with a Cy5 dye. The 29 base
pair long sequence is prone to form triple helical
structures with a third strand of DNA or RNA, making it
interesting as a potential target site for non-coding RNA
mediated regulation of gene expression. To test the
triplex forming abilities of this sequence motif in vitro, the
dsDNA was mixed with an increasing amount of single
stranded DNA that is supposed to bind, thereby forming
a triple helix. The final concentration of the labeled DNA
was 100nM. The buffer conditions were 15 mM Hepes
pH 7.4, 1 mM MgCl2 and 0.01% NP-40. After loading the
capillaries, they were incubated at 37°C for 15 min and
then measured with a laser on time of 40 sec., laser off
time of 10 sec. and a IR laser power of 15%.
Membrane Proteins
Compound binding to GPCR
Microscale thermophoresis (MST) was used to
determine whether the FC14 solubilized receptor
(vomeronasal type 1 receptor 1) could bind its ligand
myrtenal (MW 152.23). MST is the directed movement of
molecules along a spatial temperature gradient. This
movement is sensitive to changes in the hydration shell
surrounding the molecule. Ligand-binding alters this
shell in a way that measurably changes the molecules'
thermophoretic movement. MST yields results that are
comparable to SPR and other binding assays. However,
unlike SPR or other surface-based techniques, MST
does not require immobilization. The molecules are
monitored in free solution. Additionally, proteins can be
tracked by detecting the fluorescence of native
tryptophans. Coupling-chemistries or other modifications
that could potentially alter a receptors' function are thus
not necessary.
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Agonist binding to GluR2 ion channel
AMPA receptors (GluR1–4) are a subtype of the
ionotropic glutamate receptor family of ligand-gated ion
channels and have a high affinity for the full agonist
AMPA. AMPA receptors also bind and activate in
response to the nonselective, full agonists L-glutamate.
Crystallographic studies reveal that full and partial
agonists bind to the cleft of the ‘‘clamshell-shaped’’
GluR2 S1S2J ligand-binding core: The full agonists
AMPA, glutamate brings the domains of the ligandbinding core 21° closer together, relative to the apo
state. The affinity of L-Glutamate to fluorescently labeled
GluR2 was measured by MST. The affinity is in very
good agreement with literature values ((Armstrong et al,
PNAS 100, 10 (2003)))
Material was kindly provided by Prof. Dirk Trauner,
Chemical Genetics and Chemical Biology, LMU Munich
Lipids and Liposomes
Docking of DOPC vesicles
Membrane Vesicle Interaction. MST was used to monitor
the docking of two membrane vesicle populations.
Vesicles were produced by sonication and were in the
range of 30-50 nm. The binding mediating compounds
were two three heptad repeat coiled coil-forming
peptides (E and K) attached to small unilamellar vesicles
(SUV) consisting of DOPC (1,2-dioleoyl-sn-glycero-3phosphocholine). The vesicle population presenting the
E-peptide was labeled with 0.5 mol% of the fluorophor
NBD. The vesicle population labeled with K-peptide was
titrated with increasing concentration. A KD of 13 nM ± 7
was observed.
Material was kindly provided by Prof. Andreas Janshoff,
University of Göttingen, Dept. of Physical Chemistry.
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Multi Subunit Complexes
Ribosome Protein Interaction
To understand the interaction of a multimeric ribosomal
interactor with the ribosome, microscale thermophoresis
was applied. We used single cystein mutants of the
complex and coupled cystein reactive dyes for
monitoring the change in the migration in the
temperature field. Ribosomes are titrated from 2500nM
to 0.1nM. The experiments are performed in 20 mM
Hepes-HOH, pH 7,4, 100 mM KOAc, 10 mM MgOAc, 2
mM DTT and 500 nM BSA.
Results were kindly provided by Julian Deeng, AG
Beckmann, Genzentrum der LMU München
Protein Ion
Calmodulin binding to Calcium ions
MST was used to measure the specific interaction of
Ca2+-ions with fluorescently labeled calmodulin (CaM,
16.7 kDa). Upon binding to calcium, CaM undergoes a
conformational change, rearranging more than 35 water
molecules per CaM. The concentration of calcium ions
was varied from 1 nM to 100 μM while the concentration
of the protein calmodulin was kept constant at 150 nM. A
dissociation constant of KD = 2.8 ± 0.2 μM was
measured for Ca2+ binding to calmodulin. In contrast, no
binding was observed with Mg2+-ions.
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Synaptotagmin binding to Calcium ions
Karsten Meyenberg(1) and Geert van den Bogaart(2)
1 University Göttingen, Institut für Organische und
Biomolekulare Chemie, Tammannstrasse 2, D-37077
Göttingen, Germany
2 Max Planck Institute for Biophysical Chemistry,
Department of Neurobiology, Am Faßberg 11, D-37077
Göttingen, Germany
The synaptic vesicle protein synaptotagmin 1 is the main
calcium sensor of neuronal exocytosis. Calcium binds to
its cytosolic portion that consists of tandem C2-type
domains. In this work we show that Thermophoresis is a
valuable tool to measure binding of ions to proteins, with
and without the use of a fluorescent label. All protein
constructs used were from Rattus norvegicus and cloned
into the expression vector pET28a. Expression and
purification of the C2AB fragment (aa97-421) has been
described before (Stein, A., et al. 2007). Labeled protein
approach: The protein was labeled with the amine
reactive dye NT-647 according to the labeling protocol of
the respective labeling kit (NanoTemper cat#L001). A
dilution of calcium chloride starting at 20 mM in 20 mM
HEPES, 150 mM KCl at pH 7.4 was prepared. 10 µl of
the ion containing solution was mixed with 10 µl of 80
nM protein diluted in 20 mM HEPES, 150 mM KCl at pH
7.4 containing 0.5 mg/ml BSA. After mixing, the samples
were incubated for 10 minutes and filled into
hydrophobic capillaries (top graph). A similar experiment
was recently published (van den Bogaart et al. 2011)
using a cysteine labeled synaptotagmin protein. Labelfree approach (bottom graph): 10 µl of a 2 µM protein
solution was mixed with the same serial dilution of
calcium ions prepared before. As a negative control, for
both the label- and label-free approach, a serial dilution
of magnesium chloride was prepared and mixed with the
respective protein preparation. The binding of calcium
ions was observed as a clear and strong response in
MST signal, while no change in signal was observed at
increasing magnesium ion concentrations. Since
synaptotagmin-1 binds a total of 5 calcium ions, the
MST-Signal comprises of different binding events.
Therefore, the data sets are fitted with a line to guide the
eye. The dissociation constants from double digit µM to
mM are in good agreement with the literature values of
50 µM to 3 mM (Radhakrishnan et al. 2009).
Download the complete application note for further
details.
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Interaction Competition
This category refers to the use of reporter assays to
generate MST signals. In contrast to direct binding
assays, here the binding signal is generated by the
release of a molecule out of a preformed complex, upon
interaction with the titrated molecule. Either a fluorescent
or non-fluorescent molecule might be released.
Protein Compound Interaction
Compound binding to p38
Fluorescently labeled tracer molecule (Tracer199,
Invitrogen) is used at a concentration of 50nM and mixed
with a serial dilution of active p38 starting at 1µM (A).
For the experiment described here, a 50 mMTris buffer
pH 7.6 containing 150 mM NaCl, 10 mM MgCl2 and 0.05
% Tween-20 has been used. A decreasing MST signal
with increasing protein concentration (Fnorm [‰] starting
at 805 units, decreasing to 738 units) is observed with a
sigmoidal behavior that allows deducing a KD of about
80 nM. This experiment is sufficient to characterize the
interaction between Tracer and the p38 kinase (A).
Following this experiment 150 nM of p38 protein is
mixed with 25nM of Tracer199. To this stock solution a
serial dilution of the compound SB203580 (MW = 377.4
Da) starting at 4 µM is added (B). This molecule is
known to have a high affinity to the protein p38 IC50=34
nM in vitro and 600 nM in cells. After incubation of 20
minutes the MST signal of the samples is measured.
The signal shown starts at an Fnorm level of about 760
units. Thus, a significant amount of the tracer is in
complex with the protein. When increasing the
concentration of SB 203580, the MST signal increases
to about 805 units, which is exactly the signal level we
expect for free Tracer 199 thermophoresis. The signal
allows determining an IC50 of 80 nM and taking the
competition and the protein concentration into account a
dissociation constant of 20 nM in good accordance with
literature values.
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details.
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Membrane Receptor Interaction
SNARE Interactions
In eukaryotes, most intracellular membrane fusion
reactions are mediated by the interaction of
complementary SNARE proteins that are present in both
fusing membranes. The following experiment shows the
result of two different liposome populations with
compatible SNAREs incorporated in their membranes
that bind to each other, followed by membrane fusion.
One liposome population contains the neuronal SNARE
protein synaptobrevin-2 (syb-2), while the other contains
a receptor complex consisting of SNAP-25, syntaxin-1A
and a fragment of syb-2 (residues 49-96) that is labeled
with Alexa Fluor 488. Full length syb-2 binds to the
acceptor SNAREs and a cis-SNARE complex is formed.
This results in the replacement of the fluorescently
labeled syb-2 (49-96) fragment and is directly followed
by membrane fusion. Thus a signal is generated upon
binding of the two receptors. This approach has been
used instead of using a labeled liposome to separate the
receptor interaction from the following process of
liposome fusion. The result of a thermophoresis
experiment as a function of the concentration of
(unlabeled) syb-2 liposomes is shown in the figure. The
concentration of labeled acceptor SNARE liposomes has
been kept constant. An apparent dissociation constant of
about 450 nM was obtained. This binding curve shows
the dissociation of the syb-2 (49-96) fragment. Since this
dissociation is irreversible, the result reflects the
concentration of active acceptor SNAREs. The binding
curve that is obtained (in equilibrium) thus shows a
relatively strong change in the region of very high
concentrations of (unlabeled) syb-2 liposomes, whereas
at low concentrations the MST signal change is only
small because only little of the syb-2 (49-96) dissociates
of. The apparent dissociation concentration is reached
when all SNAREs are present at a molar ratio of 1:1. As
a control, plain liposomes containing no synabtobrevin
have been titrated to the labeled liposomes. As expected
no thermophoretic signal is observed. This experiment
demonstrates that even complexes with a size of
serveral 100nm can be analyzed with MST. The use of
liposomes allows to measure membrane associated
proteins and trans-membrane proteins at conditions that
are, in comparison, close to the native conditions.
Material was kindly provided byKarsten Meyenberg,
Prof. Ulf Diederichsen (Institut für Organische und
Biomolekulare Chemie, Georg-August-Universität
Göttingen) and Geert van den Bogaart and Reinhard
Jahn (Max-Planck-Institut für biophysikalische Chemie,
Göttingen).
Download the complete application note for further
details.
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Stoichiometry
This category refers to applications that determine the
number of binding sites on a molecule. These
application require a different experimental setting. Here,
the molecule which is kept constant is used at a
concentration, that is well above the dissociation
constant of the interaction. A binding partner is titrated
in. The molecular ratio, at which the saturation of binding
sites is reached yields the number of binding sites and/or
activity of the protein preparation.
Protein Compound
Determining the binding sites of streptavidin
for biotin
The interaction of fluorescently labeled streptavidin to
biotin was chosen as a model system. Streptavidin is a
tetrameric protein (MW = 53 kDa) with up to 4 high affine
binding sites for biotin. The general approach to
determine the number of binding sites with MST to
choose a concentration of the constant binding partner
that is well above the KD of the interaction. When the not
labeled binding partner is titrated to the labeled
streptavidin it is almost completely bound up to the point,
where all binding sites are occupied. In this experiment,
the 4 binding sites were quickly saturated, by combining
the interaction partners at concentrations of 200 nM
Streptavidin (i.e. higher than the KD), resulting in a
saturation curve that shows a characteristic kink, when
saturation is reached. The molecular ratio of titrant and
labeled molecule directly yield the number of binding
sites (and activity of protein). In this experiment, a value
of "3,75+/-0.2 active sites" was measured for biotin
binding to streptavidin. The decimal value was due to
protein activity effects and slightly below the theoretical
value of 4 active sites, achievable only when the protein
preparation is 100% active. The experiment was
repeated with biotin coupled to ssDNA molecules of
different size and as expected, the active binding sites
value decreased with increasing DNA length, nicely
demonstrating the steric hindrance caused by the
ssDNA flag attached to biotin.
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Binding Energetics
This category refers to applications that allow to
measure the thermodynamics of an interaction. The
enthalpy and entropy of an interaction can be
determined, by measuring the affinity at different
ambient/sample mount temperatures. The resulting KD
temperature dependencies are analyzed using the van't
Hoff approach.
Protein Compound
Thermodynamics of p38 binding to Small
Molecule Inhibitor
In this application note we demonstrate that it is possible
to gather this thermodynamic information by the use of
microscale thermophoresis (MST), taking full advantage
of its unique benefits such as tiny sample consumption,
and a temperature controlled sample mount. Using this
methodology it is feasible to gather valuable
thermodynamic data early on in the drug discovery
process. p38 is a serine/threonine protein kinase in the
mitogen-activated protein kinase (MAPK) family. There
are four isoforms of p38 (p38α, p38β, p38γ, and p38δ),
and p38α is considered as the key isoform involved in
modulating inflammatory response in rheumatoid arthritis
and inflammatory pain (Dominuez et al., Curr Opin Drug
Discov Devel, 2005). Initially the binding of Tracer199 to
the inactive form of p38α was confirmed at room
temperature and standard conditions. The Kd was
determined to be 5,4 ± 0,3nM, which is in excellent
agreement with the data published by Invitrogen on their
website (Invitrogen, Catalog Number: PV5830).Upon
increasing the temperature, the inflection point of the
curve is gradually moved to the right, as the apparent
affinity decreases. Over a temperature range of 20°C the
Kd shifts from 5,4nM to 200nM, as shown in the figures
below: In the van 't Hoff analysis, the natural logarithm of
the equilibrium constant Kd is plotted against the inverse
Temperature 1/T, whereas T is the absolute temperature
in Kelvin. Upon this transformation a linear plot is
achieved, where the slope of the line yields ∆Hᶱ and the
intercept is -∆Sᶱ/R. The derived ∆Hᶱ is -22,4 kcal/mol
and ∆Sᶱ equates to 0,05 kcal/mol.
Download the complete application note for further
21
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5.Publications
2011
Signaling by the Matrix Proteoglycan Decorin
Controls Inflammation and Cancer Through PDCD4
and MicroRNA-21
Rosetta Merline, Kristin Moreth, Janet Beckmann,
Madalina V. Nastase, Jinyang Zeng-Brouwers, Jose
Guilherme Tralhao, Patricia Lemarchand, Josef
Pfeilschifter, Roland M. Schaefer, Renato V. Iozzo, and
Liliana Schaefer
Science Signal. : DOI: 10.1126/scisignal.2001868
(2011)
Vaccines Against Drug Abuse
X Y Shen, F M Orson, and T R Kosten
Clinical Pharmacology & Therapeutics
:doi:10.1038/clpt.2011.281 (2011)
Structure and function analyses of the purified
GPCR human vomeronasal type 1 receptor 1
Karolina Corin, Philipp Baaske, Sandra Geissler,
Christoph J. Wienken, Stefan Duhr, Dieter Braun,
Shuguang Zhang
Scientific Reports 1 (172). doi:10.1038/srep00172
(2011)
Microscale Thermophoresis as a Sensitive Method
to Quantify Protein: Nucleic Acid Interactions in
Solution
Karina Zillner, Moran Jerabek-Willemsen, Stefan Duhr,
Dieter Braun, Gernot Längst, Philipp Baaske
Springer Protocols, Methods in Molecular Biology:
10.1007/978-1-61779-424-7_18 (2011)
Designer Lipid-Like Peptides: A Class of Detergents
for Studying Functional Olfactory Receptors Using
Commercial Cell-Free Systems
Karolina Corin, Philipp Baaske, Deepali B. Ravel,
Junyao Song, Emily Brown, Xiaoqiang Wang, Christoph
J. Wienken, Moran Jerabek-Willemsen, Stefan Duhr,
Yuan Luo, Dieter Braun, Shuguang Zhang
PLoS ONE 6(11): e25067.
doi:10.1371/journal.pone.0025067 (2011)
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2011 continued
A Robust and Rapid Method of Producing Soluble,
Stable, and Functional G-Protein Coupled Receptors
Karolina Corin, Philipp Baaske, Deepali B. Ravel,
Junyao Song, Emily Brown, Xiaoqiang Wang, Sandra
Geissler, Christoph J. Wienken, Moran JerabekWillemsen, Stefan Duhr, Dieter Braun, Shuguang Zhang
PLoS ONE 6(10): e23036,
doi:10.1371/journal.pone.0023036 (2011)
Saccharomyces cerevisiae Ngl3p is an active 3′–5′
exonuclease with a specificity towards poly-A RNA
reminiscent of cellular deadenylases
Ane Feddersen, Emil Dedic, Esben G. Poulsen, Manfred
Schmid, Lan Bich Van, Torben Heick Jensen and Ditlev
E. Brodersen
Nucleic Acids Research, DOI: 10.1093/nar/gkr782
(2011)
Molecular Interaction Studies Using Microscale
Thermophoresis
Moran Jerabek-Willemsen, Christoph J. Wienken, Dieter
Braun, Philipp Baaske and Stefan Duhr
ASSAY and Drug Development Technologies,
DOI:10.1089/adt.2011.0380 (2011)
Atomic resolution structure of EhpR: phenazine
resistance in Enterobacter agglomerans Eh1087
follows principles of bleomycin / mitomycin C
resistance in other bacteria
Shen Yu, Allegra Vit, Sean Devenish, H KHRIS
Mahanty, Aymelt Itzen, Roger S Goody and Wulf
Blankenfeldt
BMC Structural Biology, DOI:10.1186/1472-6807-1133 (2011)
Synaptotagmin-1 may be a distance regulator acting
upstream of SNARE nucleation
Geert van den Bogaart, Shashi Thutupalli, Jelger H
Risselada, Karsten Meyenberg, Matthew Holt, Dietmar
Riedel, Ulf Diederichsen, Stephan Herminghaus, Helmut
Grubmüller and Reinhard Jahn
Nature Structural & Molecular Biology,
doi:10.1038/nsmb.2061 (2011)
NEMO interaction with linear and K63 ubiquitin
chains contributes to NF-kB activation
Kamyar Hadian, Richard A. Griesbach, Scarlett
Dornauer, Tim M. Wanger, Daniel Nagel, Moritz
Metlitzky, Wolfgang Beisker, Marc Schmidt-Supprian
and Daniel Krappmann
JBC, DOI: 10.1074/jbc.M111.23316 (2011)
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2011 continued
A comparative study of fragment screening methods
on the p38a kinase: new methods, new insights
Pollak et al.
J Comput Aided Mol Des, DOI 10.1007/s10822-0119454-9 (2011)
Peptide surfactants for cell-free production of
functional G protein-coupled receptors
Xiaoqiang Wang, Karolina Corin, Philipp Baaske,
Christoph J. Wienken, Moran Jerabek-Willemsen, Stefan
Duhr, Dieter Braun and Shuguang Zhang
PNAS, DOI: 10.1073/pnas.1018185108 (2011)
Thermophoretic melting curves quantify the
conformation and stability of RNA and DNA
Christoph J. Wienken, Philipp Baaske, Stefan Duhr and
Dieter Braun
Nucleic Acids Research, DOI: 10.1093/nar/gkr035
(2011)
Investigating a macromolecular complex: The toolkit
of methods
Anastassis Perrakis,
Journal of Structural Biology, Volume 175, Issue 2,
August 2011, Pages 106-112
2010 and earlier
Protein Binding Assays in Biological Liquids using
Microscale Thermophoresis
Christoph J. Wienken, Philipp Baaske, Ulrich Rothbauer,
Dieter Braun and Stefan Duhr
Nature Communications, DOI: 10.1038/ncomms1093
(2010)
Quantum Dots Modulate Leukocyte Adhesion and
Transmigration Depending on their Surface
Modification
M. Rehberg , M. Praetner , C. F. Leite , C. A. Reichel , P.
Bihari , K. Mildner , S. Duhr , D. Zeuschner and F.
Krombach
Nano Letters, 10(9), 3656-3664 (2010)
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2010 and earlier, continued
Targeting multi-functional proteins by virtual
screening: structurally diverse cytohesin inhibitors
with differential biological functions
Jürgen Bajorath , Dagmar Stumpfe , Anke Bill , Nina
Novak , Gerrit Loch , Heike Blockus , Hanna Claudia
Geppert , Thomas Becker , Michael Hoch , Michael
Famulok , Waldemar Kolanus and Anton Schmitz
ACS Chemical Biology, DOI: 10.1021/cb100171c
(2010)
Optical Thermophoresis for Quantifying the Buffer
Dependence of Aptamer Binding
Philipp Baaske, Christoph J. Wienken, Philipp Reineck,
Stefan Duhr and Dieter Braun
Angewandte Chemie International Edition, 49, 22382241 (2010)
Thermophoresis of Single Stranded DNA
Philipp Reineck, Christoph J. Wienken and Dieter Braun
Electrophoresis 31, 279–286 (2010)
Optisch erzeugte Thermophorese für die Bioanalytik
Philipp Baaske, Christoph Wienken, Stefan Duhr
BioPhotonik 2009 (Rubrik Laser in Medizin und
Biologie)
Melting curve analysis in a snapshot
Philipp Baaske, Stefan Duhr and Dieter Braun
Applied Physics Letters 91, 133901 (2007)
Why molecules move along a temperature gradient
Stefan Duhr and Dieter Braun
PNAS 103, 19678–19682 (2006)
V35
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Contact
NanoTemper Technologies GmbH
Floessergasse 4
81369 Munich
Germany
Tel.: +49 (0)89 4522895 0
Fax: +49 (0)89 4522895 60
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