Microscale Thermophoresis Technology and Applications

1

www.nanotemper.de

Contents

1. Technology

2. Technology Platform

3. Handling

4. Applications

5. Publications

1

www.nanotemper.de

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-Maximilians-University 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 IR-Laser 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.

Microscale Thermophoresis

1. Technology

2

www.nanotemper.de

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.

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. 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.

Monolith Series Instruments

2. Technology Platform

3

www.nanotemper.de

Key Benefits

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.

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:

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

Unmatched Sensitivity

Unique performance

Close-to-native conditions

4

www.nanotemper.de

Monolith NT.115

LED 1 /nm

LED 2 /nm

Blue Dyes Green Dyes Red Dyes

NT.115 Blue/Green

Ex:470

Em:520

Ex:550

Em:600

FITC/FAM/GFP/YFP Cy3/RFP/mCherry no detection

NT.115 Blue/Red

Ex:470

Em:520

Ex:625

Em:680

FITC/FAM/GFP/YFP no detection Cy5/Alexa647

NT.115 Green/Red

Ex:520

Em:570

Ex:625

Em:680

YFP

Cy3/RFP Cy5/Alexa647

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. 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.

Capillary format

Requires little sample material

Dedicated Data Acquisition and Analysis Software

Monolith Models

Monolith NT.115

5

www.nanotemper.de

Monolith NT.LabelFree

LED 1 /nm

Molecules (examples)

NT.Label Free

Ex:280

Em:360

Proteins containing Tryptophane 2-Aminopurin

8-vinyl-deoxyadenosine BIRB-796

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, 2-Aminopurin, 8-vinyl-deoxyadenosine, etc)

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. 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.

Monolith NT.LabelFree

Capillaries

Monolith Consumables

6

www.nanotemper.de

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.

Our Assay Development and Control Kits allow you to

get started with MST quickly and train new lab members.

Assay Development and Control Kits

Labeling Kits

7

www.nanotemper.de

Microscale Thermophoresis is easy to handle and

involves the following steps:

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.

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.

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.

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.

Labeling (only NT.115)

3. Handling

Mixing the Reaction

Transfer in Capillary

Measurement and Data Analysis

8

www.nanotemper.de

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.

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)

Interaction Direct

Protein-Protein Interaction

GFP binding to GFP-binding Protein

4.Applications

Protein Kinase A interaction with PKI

9

www.nanotemper.de

Stephen H. McLaughlin

MRC Laboratory for Molecular Biology, Cambridge, UK

For proper folding, many proteins involved in signal-transduction 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.

p38 is a serine/threonine protein kinase in the mitogen-

activated 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.

Small Molecules

17-DMAG binding to Hsp90 Protein

Binding of Small Molecules to labeled p38

10

www.nanotemper.de

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 ligand-

binding 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

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.

Agonist binding to GluR2 ion channel

Binding of Triostin Analogues to DNA

11

www.nanotemper.de

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 GST-

AT1+2 has a five times higher affinity (EC50=4 µM) to

the DNA than GST-AT1 (EC50=20 µM).

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).

Protein Nucleic Acid

HMG protein binding to dsDNA

Aptamer Interaction with Thrombin

12

www.nanotemper.de

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

Protein Ku70/Ku90 interaction with dsDNA

13

www.nanotemper.de

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, CAF-1 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 WD-repeat proteins. Here we checked the interaction of p48 with Histone H3 derived peptides. Fig. 2 shows the resulting binding curve for the H3NS peptide - NT647-labeled p48 interaction with a calculated Kd of 15.76 ± 2.18µM.

Download the complete application note for further details.

Binding of Histone peptides to Chromatin assembly

factor I (CAF-I) p48 subunit

Protein Peptide

14

www.nanotemper.de

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%.

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.

DNA Triple-Helix Formation

Nucleic Acids

Compound binding to GPCR

Membrane Proteins

15

www.nanotemper.de

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 ligand-

binding 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

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-3-phosphocholine). 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.

Agonist binding to GluR2 ion channel

Lipids and Liposomes

Docking of DOPC vesicles

16

www.nanotemper.de

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

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.

Ribosome Protein Interaction

Multi Subunit Complexes

Calmodulin binding to Calcium ions

Protein Ion

17

www.nanotemper.de

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. Label-free 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.

Synaptotagmin binding to Calcium ions

18

www.nanotemper.de

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.

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.

Download the complete application note for further details.

Interaction Competition

Protein Compound Interaction

Compound binding to p38

19

www.nanotemper.de

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.

Membrane Receptor Interaction

SNARE Interactions

20

www.nanotemper.de

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.

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.

Stoichiometry

Protein Compound

Determining the binding sites of streptavidin for biotin

21

www.nanotemper.de

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.

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

Binding Energetics

Protein Compound

Thermodynamics of p38 binding to Small

Molecule Inhibitor

22

www.nanotemper.de

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)

5.Publications

2011

23

www.nanotemper.de

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 Jerabek-Willemsen, 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:10.1186/1472-6807-11-33 (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)

2011 continued

24

www.nanotemper.de

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/s10822-011-9454-9 (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

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)

2011 continued

2010 and earlier

25

www.nanotemper.de

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, 2238-2241 (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)

2010 and earlier, continued

V35

www.nanotemper.de

Contact

NanoTemper Technologies GmbH Floessergasse 4

81369 Munich

Germany

Tel.: +49 (0)89 4522895 0

Fax: +49 (0)89 4522895 60

info@nanotemper.de

http://www.nanotemper.de