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MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
微量热泳动法研究大真核生物蛋白复合物中蛋白质 - 肽相互作用   

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The Plant Cell
Apr 2017

Abstract

Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides is thus an important task in systems biology, cell biology, biochemistry, and drug design. However, this task is often hampered by the drawbacks of classical biophysical methods for analysis of molecular interactions like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), which require immobilization of the interaction partners or very high sample concentrations. MicroScale Thermophoresis (MST) is an innovative method that offers the possibility to determine the important parameters of a molecular interaction, such as dissociation constant, stoichiometry, and thermodynamics. Moreover, it does so in a rapid and precise manner, with free choice of buffers or biological liquids, no need for sample immobilization, and very low sample consumption. Here we describe two MST assays in detail, which analyze (i) the interactions between certain peptide stretches of the eukaryotic RNA polymerase II and a protein subunit of the eukaryotic transcription elongation complex and (ii) interactions between N-terminal histone tail peptides and epigenetic reader proteins. These experiments show that MST is able to characterize protein-peptide interactions that are triggered by only minor changes in the peptide, for example, only one phosphorylation at a specific serine residue.

Keywords: MicroScale Thermophoresis (微量热泳), Molecular interactions (分子相互作用), Binding affinity (结合亲和力), Binding parameters (结合参数), Protein-peptide interactions (蛋白质 - 肽相互作用), Histones (组蛋白), Epigenetics (表观遗传学), RNA polymerase (RNA聚合酶)

Background

Biological Background: Protein-protein interactions (PPIs) are essential for almost all cellular processes, ranging from DNA replication, transcription, and translation over the formation of metabolic enzyme complexes and large cellular machineries, to the sensing and relay of biological signals. Given the physiological importance of PPIs, protein complexes are considered high-value targets in drug development (Milroy et al., 2014; Nevola and Giralt, 2015). However, traditional, small-molecule based targeting approaches often fail to produce potent inhibitors, mostly due to the large and often flat protein interfaces (Sperandio et al., 2010). Alternative strategies involve peptide-based inhibitors that often mimic part of the binding surface of an interaction partner (Boersma et al., 2012; Azzarito et al., 2013; Arkin et al., 2014; Pelay-Gimeno et al., 2015). Such interactions between proteins and peptides of typically 5-20 amino acids are frequently found in nature, for example with antibodies, kinases, phosphatases, MHC proteins, or epigenetic reader proteins (Stanfield and Wilson, 1995). Moreover, the specific targeting of PPIs with small peptides can help to elucidate composition and function of large and sophisticated protein complexes like the eukaryotic RNA polymerase II elongation complex (Antosz et al., 2017) or the specificity of epigenetic reader proteins (Josling et al., 2015).

Technical Background: In order to precisely and comprehensively characterize protein-protein and protein-peptide interactions with respect to affinity, kinetics, and thermodynamics, an array of biophysical techniques and methods have been developed. Well established technologies are isothermal titration calorimetry (ITC) (Pierce et al., 1999; Ghai et al., 2012), surface plasmon resonance (SPR)-based assays (Pattnaik, 2005; Ishii et al., 2013), native mass spectrometry (Heck, 2008; Yan et al., 2017), biolayer interferometry (Abdiche et al., 2008; Concepcion et al., 2009), static or dynamic light scattering (Wen et al., 1996; Kameyama and Minton, 2006; Hanlon et al., 2010), as well as fluorescence spectroscopy (Lundblad et al., 1996; Lin et al., 2014; Raines, 2015). Although some of these methods uniquely provide specific information like on- and off-rates or the precise complex stoichiometry, they are often lab-intense and complex, are often problematic due to high sample consumption, low-sensitivity, surface immobilization, mass transport limitations, as well as buffer restrictions or fail to quantify interactions involving small peptides. The innovative MicroScale Thermophoresis technology (MST) is a powerful technique that overcomes such limitations and allows for the fast, precise, cost-efficient, and quality-controlled characterization of molecular interactions with very low sample consumption, no need of sample immobilization, and a free choice of buffers or bioliquids. MST enables researchers to determine the important parameters of molecular interactions, such as binding affinity (from pico-molar o milli-molar), binding stoichiometry, and interaction thermodynamics (Wienken et al., 2010; Jerabek-Willemsen et al., 2011). Importantly, MST does not impose any limitations regarding molecular weight of the interacting molecules and thus quantifying interactions between proteins of several hundred kDa in size and short peptides with molecular weights below 1 kDa is straightforward and precise (Antosz et al., 2017).

The physical phenomenon of thermophoresis: The physical phenomenon of ‘thermophoresis’ describes the movement of molecules along a temperature gradient. This movement is dependent on the size, charge, and hydration shell of the molecules (Braun and Libchaber, 2002; Duhr and Braun, 2006). Interactions between a protein and a ligand (other protein, peptide, DNA, RNA, small molecule) alter at least one of these parameters resulting in a different thermophoretic mobility along the temperature gradient (Jerabek-Willemsen et al., 2014). Thus interactions between large proteins and small ligands, such as short peptides, which only entail negligible changes in size and charge, will significantly influence the thermophoresis due to changes in the hydration shell.

MicroScale Thermophoresis: MicroScale Thermophoresis is an innovative technology that utilizes the thermophoretic principle to study molecular interactions in solution (Duhr et al., 2004; Baaske et al., 2010; Jerabek-Willemsen et al., 2011). The thermophoretic movement of molecules is induced by generating a microscopic temperature gradient inside a very thin glass capillary using an infrared (IR) laser. The corresponding technical setup of an MST device (Monolith NT.115, NanoTemper Technologies GmbH, Munich, Germany) is shown in Figure 1A. This gradient is focused in a diameter of around 50 µM and comprises a temperature difference ∆T of 2-6 °C. In the same area, the thermophoretic movement of the molecules is tracked by fluorescence optics using either the intrinsic fluorescence of tryptophans in proteins or peptides (Seidel et al., 2012) or the fluorescence signal of an extrinsic fluorophore that is coupled to one of the interacting partners (Schubert et al., 2012; Zillner et al., 2012). Upon heating, molecules either deplete or accumulate in the center of the temperature gradient and the fluorescence optics, which can be quantified by the Soret coefficient ST, with chot and ccold representing the concentration in the heated and non-heated regions of the capillary and ∆T the temperature difference along the gradient (Duhr and Braun, 2006):



A typical MST measurement is depicted in Figure 1B. In the beginning, the fluorescence in the capillary is measured at the fixed starting temperature for usually five seconds. Then the temperature gradient is induced by the IR laser, which results in a steep drop of the measured fluorescence signal, the so-called temperature- or T-jump. This signal jump results from temperature-dependent changes in the quantum yield of the used fluorophore. After that, a slower, thermophoresis-driven depletion of fluorescent molecules in the optical focus corresponding to the Soret coefficient results in an exponential decrease of the fluorescence signal. After a measurement time of typically 20-30 sec, the IR laser is turned off again, which results in a thermophoretic back-diffusion of fluorescent molecules into the optical focus and a concomitant reverse T-jump.

In order to determine the equilibrium dissociation constant (KD) of the complex under study, a series of MST measurements with a fixed amount of fluorescent binding partner and increasing amounts of non-fluorescent partner are recorded (Figure 1C). The 16 MST traces are normalized with respect to their initial fluorescence. The concentration range of the non-fluorescent partner is chosen in a way that the lowest concentration results in a practically fully ‘unbound’ fluorescent partner and the highest concentration results in a fully ‘bound’ fluorescent partner. Usually 16 dilutions of the non-fluorescent partner are prepared ranging from about 10-fold above to the 10-fold below the projected KD, supplemented with the same amount of fluorescent partner and loaded into 16 individual capillaries. The concentration of the fluorescent partner is usually kept below the projected KD in order to allow for a precise determination of the true equilibrium affinity. The differences between the ‘cold’ and ‘hot’ states of each of the 16 MST traces is then used to determine the change in fluorescence for each trace:



Plotting these values against the ligand concentration finally results in a typical binding isotherm, which yields the KD value of the interaction (Figure 1D).


Figure 1. MicroScale Thermophoresis. A. Technical setup of an MST device. The thermophoretic movement of molecules inside the glass capillary is induced by an infrared laser that is focused on a 50 µM wide area. In the same area fluorescence emission is used to track this movement. In total, 16 capillaries can be analyzed subsequently in one device. B. An example of MST trace. After an initial delay of five seconds, the IR laser is turned on to establish a temperature gradient. Following the T-jump phase, the thermophoretic movement leads to an exponential decrease of fluorescent molecules in the optics focus. After a measurement time of typically 20-30 sec, the laser is turned off. C. Combined traces of a typical MST experiment in which 16 capillaries are analyzed that contain the same concentration of the fluorescent interaction partner but increasing concentrations of the non-fluorescent partner. All traces are normalized to the same initial relative fluorescence value of 1. D. Final binding isotherm resulting from plotting the difference in normalized fluorescence against the concentration of the non-fluorescent binding partner (ligand). Figure modified from Entzian and Schubert, 2016; originally kindly provided by NanoTemper Technologies, Munich.

Advantages and drawbacks of MST: MST offers several integral advantages over other biophysical methods for characterizing protein-protein and protein-peptide interactions. First, the MST traces and lateral scans of the filled capillaries provide a straight-forward sample quality control as they allow for the easy detection of aggregation, precipitation, and adsorption effects. This enables the researcher to quickly alter and optimize technical and buffer conditions to increase sample stability and data quality. Importantly, these controls take less than two minutes for 16 capillaries. Also the full required measurement to get an equilibrium KD takes less than 15 min. Moreover, MST is immobilization-free and thus allows for the determination of affinities in practically all buffers and even in complex bioliquids like lysates and sera (Wienken et al., 2010; Seidel et al., 2013). Also, MST excels by very low sample consumption, which can be a crucial issue when working with hard-to-produce eukaryotic proteins. Usually, the fluorescent interaction partner is assayed at very low nanomolar concentrations (5-20 nM) and the non-fluorescent partner usually–depending on the KD–in the nanomolar to low micromolar range. Finally, the high dynamic range of detectable affinities (pM to mM) and non-existing size limitations allow for the characterization of a wide variety of different molecular interactions involving proteins, peptides, small molecules, DNA, and RNA, just to name a few. However, MST does not allow for determining on and off-rates of molecular interactions. Nevertheless, binding parameters derived from MST experiments agree well with established state-of-the-art methods like SPR or ITC (Ramakrishnan et al., 2012; Chen et al., 2015; Stoltenburg et al., 2015; Wan et al., 2015; Harazi et al., 2017). Finally, it has to be stated that for most MST applications one interaction partner has to be modified with a fluorophore. Thus, if neither a peptide, nor a protein can be labeled with a fluorophore (e.g., because neither contains lysine or cysteine residues for fluorophore coupling), standard MST cannot be applied. In such cases, however, label-free MST analysis can be an alternative, due to the read-out of intrinsic tyrosine and tryptophan fluorescence of the protein or the peptide.

Materials and Reagents

  1. Low volume, low binding reaction tubes (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 90410 )
  2. Eppendorf tubes (1.5 ml) (Sigma-Aldrich, catalog number: T9661)
    Manufacturer: Eppendorf, catalog number: 022363204 .
  3. PCR tubes (200 µl, NanoTemper Technologies GmbH, Munich, Germany)
  4. Pipette tips (10 μl, 100 μl, 1,000 μl) (STARLAB INTERNATIONAL, catalog number: S1111 )
  5. FITC-labelled peptides of the CTD of the A. thaliana RNA polymerase II (Biomatik, Wilmington, Delaware, USA)
    Note: ‘pS’ denotes a phosphorylated serine; peptides are labeled with Fluorescein isothiocyanate [FITC, CAS number: 27072-45-3] at the N-terminus.
    1. CTD-noP (sequence: PSYSPTSPSYSP)
    2. CTD-Ser2P (sequence: TSPSY(pS)PTSPSY)
    3. CTD-Ser5P (sequence: SYSPT(pS)PSYSPT)
  6. Protein SPT6L (Phe1218-Asp1412)
    Note: Expressed in Escherichia coli with a GST-tag and purified by glutathione-sepharose affinity chromatography as described in (Kammel et al., 2013).
  7. Protein HP1 from Plasmodium falciparum (P. falciparum)
    Note: Expressed in E. coli BL21-CodonPlus(DE3)-RIL cells with a hexahistidine tag and purified by Ni2+-IMAC chromatography (Josling et al., 2015).
  8. Protein HP1 from Drosophila melanogaster (D. melanogaster)
    Note: Expressed in E. coli BL21-CodonPlus(DE3)-RIL cells with a hexahistidine tag and purified by Ni2+-IMAC chromatography (Josling et al., 2015).
  9. Histone peptides
    All histone peptides were purchased from ANASPEC, Kaneka Eurogentec (Liege, Belgium)
  10. Pluronic® F-127 (CAS number: 9003-11-6) (Sigma-Aldrich, catalog number: P2443 )
  11. Sodium dodecyl sulfate, sodium salt (SDS; CAS number: 151-21-3) (Sigma-Aldrich, catalog number: L3771 )
  12. Sodium phosphate (CAS number: 7601-54-9) (Sigma-Aldrich, catalog number: 342483 )
  13. Ethylenediaminetetraacetic acid (EDTA, CAS number: 60-00-4) (Sigma-Aldrich, catalog number: E9884 )
  14. DL-Dithiothreitol (DTT, CAS number: 3483-12-3) (Sigma-Aldrich, catalog number: 43815 )
  15. Phenylmethanesulfonyl fluoride (PMSF, CAS number: 329-98-6) (Sigma-Aldrich, catalog number: 78830 )
  16. Tris(hydroxymethyl)aminomethane (Tris, CAS number: 77-86-1) (Sigma-Aldrich, catalog number: T1503 )
  17. Sodium chloride (NaCl, CAS number: 7647-14-5) (Sigma-Aldrich, catalog number: S7653 )
  18. Magnesium chloride (MgCl2, CAS number: 7786-30-3) (Sigma-Aldrich, catalog number: M8266 )
  19. Tween® 20 (CAS number: 9005-64-5) (Sigma-Aldrich, catalog number: P1379 )
  20. NaP buffer (see Recipes)
  21. MST-T buffer (see Recipes)

Equipment

  1. Pipettes (Eppendorf Reference® 20, 100, 200, 1,000) (Eppendorf, catalog numbers: 4920000032 , 4920000059 , 4920000067 , 4920000083 )
  2. MiscroScale Thermophoresis instrument Monolith NT.115 (NanoTemper Technologies, model: Monolith NT.115 )
  3. MiscroScale Thermophoresis instrument Monolith NT.LabelFree (NanoTemper Technologies, model: Monolith NT.LabelFree )
  4. Monolith NTTM capillaries ‘Premium coated’ (NanoTemper Technologies, Munich, Germany)
  5. Incubator (Eppendorf, model: ThermoMixer® comfort , catalog number: 5360000011)
  6. Centrifuge (Eppendorf, model: 5424 R , catalog number: 5404000219)

Software

  1. MO.Control (NanoTemper Technologies, Munich, Germany)
  2. MO.Affinity Analysis (NanoTemper Technologies, Munich, Germany)

Procedure

  1. Solution preparation
    Notes: Buffers
    1. For the interactions between SPT6L(Phe1218-Asp1412) and the CTD peptides of the A. thaliana RNA polymerase II, the assay buffer is NaP buffer (see Recipes).
    2. For the interactions between the HP1 proteins and the histone peptides, the assay buffer is MST-T buffer (see Recipes).

    1. Preparation of the working solutions of the targets
      1. Peptides CTD-noP, CDT-Ser2P, and CDT-Ser5P: These peptides serve as targets for binding of SPT6L(Phe1218-Asp1412).
        1. Follow the manufacturers’ instructions and dissolve the peptides.
        2. Prepare the target working solution by diluting the peptide stocks to 80 nM with MST-T buffer.
      2. HP1 proteins from P. falciparum and D. melanogaster. These proteins serve as the targets for binding of the different histone peptides. Labeling of the targets is not necessary in this context, because their intrinsic tryptophan fluorescence can be used with the Monolith NT.LabelFree device.
        Prepare the target working solution by diluting the protein stocks to 3.5 µM in MST-T buffer.
    2. Preparation of the working solution of the ligand
      1. SPT6L(Phe1218-Asp1412) protein: This protein serves as the ligand for binding of the CTD-peptides.
        1. Dilute the ligand stock with MST-T buffer to 4.3 mM. Ideally, the concentration of the working solution of the ligand should be around 50-times higher than the projected KD value.
          Note: Centrifugation of the ligand stock for 5 min at 14,000 x g at 4 °C may help remove aggregates. Low volume, low binding reaction tubes (e.g., Thermo Fisher Scientific) are recommended to avoid adsorption of molecules to the tube walls.
        2. When preparing the ligand working solution it has to be considered that–depending on the dilution factor–a certain amount of ligand stock buffer can still be present in the working solution. If this exceeds 1%, we recommend adjusting the final MST buffer accordingly.
      2. Histone peptides: These peptides serve as the ligands for the measurements with the two HP1 protein targets.
        1. Follow the manufacturers’ instructions and dissolve the peptides.
        2. Prepare the ligand working stock by diluting the peptide stocks to 20 mM in NaP buffer.
    3. Preparation of the ligand dilution series
      1. The ligand is diluted in 16 serial steps in 200 µl PCR tubes. Dilute the ligand by 50% (1:1 dilution) in each of the sixteen steps.
        Note: The concentration finder tool implemented in the control software simulates binding data and helps with finding the right concentration range for the dilution series.
      2. Add 10 µl of assay buffer into PCR tubes 2 to 16.
      3. Fill 20 µl of the ligand working solution in PCR tube 1.
      4. Transfer 10 µl from PCR tube 1 into PCR tube 2 and mix properly by pipetting up and down several times.
        Note: Avoid vortexing or heavy shaking in order to prevent protein denaturation.
      5. Transfer 10 µl from PCR tube 2 into PCR tube 3 and mix. Repeat this process for the remaining dilution steps.
      6. Discard the excess 10 µl from the last tube. All 16 PCR tubes should now contain a volume of 10 µl.
        Note: It is important to avoid any buffer dilution effects. All 16 PCR tubes should contain the exact same buffer composition. If the ligand stock solution is in a different buffer than the MST assay buffer, the best practice is to prepare an aliquot of the MST assay buffer that features the exact same composition as the buffer of the ligand working solution. This buffer is then used for pipetting the serial dilution series. For example, if the ligand stock solution is in buffer A + 2 mM DTT and is diluted 10-fold in buffer A to yield the ligand working solution, the DTT concentration in the working solution is 0.2 mM. Thus use buffer A + 0.2 mM DTT for preparing the serial dilution series.
    4. Preparation of the final MST mix
      1. Although a volume of 4 µl is sufficient to fill the MST capillaries, it is advised to prepare at least 20 µl of the final MST mix in order to minimize pipetting errors.
      2. Add 10 µl of the target working solution to each 10 µl ligand dilution step and mix properly by pipetting up and down several times.
        Note: Avoid vortexing or heavy shaking in order to prevent protein denaturation.
      3. Incubate at room temperature for five minutes to achieve binding equilibrium. Longer incubation times or different incubation temperatures may be required depending on the specific targets and ligands. The specific incubation time and temperature have to be chosen with a priori knowledge about the interaction to be analyzed. As a general suggestion, we recommend to incubate for between 5 and 20 min at room temperature.
      4. Fill 16 capillaries with the 16 MST mixes by dipping the capillaries into the sample.
        Note: Do not touch the capillaries in the middle section where the optical measurement will take place.
      5. Place the capillaries onto the capillary tray and start the MST device.

  2. MST measurement
    1. Starting the MST device
      1. Start the MST control software and adjust the desired temperature by enabling temperature control. Usually measurements are performed at 25 °C. Wait for the temperature to reach the predefined value.
        Note: MST instruments can be temperature-controlled from 22 to 45 °C.
      2. Place the capillary tray in the MST device.
      3. Set the LED channel (fluorescence excitation) to ‘blue’ for FITC fluorophore labels and set the LED power to gain a fluorescence signal of 300 to 1,000 units at an MST device with a standard sensor and 6,000 to 18,000 on a device with a high-sensitivity sensor.
        Note: Other fluorescent labels may require the ‘red’ LED setting. Check the excitation wavelengths of the used fluorophore.
    2. Capillary scan
      1. Perform a capillary scan in order to check different quality aspects of the sample.
      2. Check the capillary scan to see if the maximum fluorescence signal obtained falls in the ranges described above.
      3. Check the capillary scan for sticking effects of labeled target to the glass walls of the capillaries. This leads to U-shaped or flattened peaks.
      4. Check the capillary scan for pipetting errors. These lead to inconsistent fluorescence values across the 16 MST mixes.
      5. Check the capillary scan for ligand-dependent fluorescence enhancement or quenching effects. These lead to increasing or decreasing fluorescence values with increasing ligand concentration in the 16 MST mixes.
    3. MST measurement
      1. Before starting an MST measurement, make sure to exclude any sticking effects, pipetting errors, enhancing/quenching effects, and ensure a sufficient fluorescence signal.
      2. Assign each of the 16 MST mixes with the respective final ligand concentration in the control software. In newer control software versions, only the highest ligand concentration (usually in MST mix 1) is set and the dilution type (e.g., 1:1 or 1:2) is chosen.
        Note: The ligand concentration that has to be entered at this point is half the concentration of the ligand dilution series described above, because the dilution samples were mixed 1:1 with target solution.
      3. Enter the fixed concentration of the fluorescently labeled target.
        Note: This concentration is also half the concentration of the target working solution due to the preparation of the MST mixes described above.
      4. For most applications, the default settings (initial fluorescence for 5 sec, recording of thermophoresis for 30 sec, and after-thermophoresis fluorescence for a further 5 sec) are sufficient.
      5. Adjust the MST-power to 20%.
        Notes:
        1. In order to receive the best signal-to-noise ratio and to avoid unspecific effects, a laser power of 20-40% is recommended. In some cases, a higher laser power may be required to get good separation of unbound and bound molecules.
        2. In newer control software versions only the MST power settings ‘low’, ‘medium’, and ‘high’ are available. ‘Medium’ is recommended for most applications.
      6. Enter a destination folder path where the experiment file will be saved. The experiment will be saved as a .ntp file.
      7. Start the MST measurement. The measurement will take between 10-15 min, depending on the times set.
      8. Repeat the MST measurement at least twice for a more reliable determination of the equilibrium binding affinity.
        Note: In order to test the technical reproducibility of the measurement, the same capillaries can be used for several MST measurements.

Data analysis

  1. Data analysis
    1. Start the MST analysis software (MO.Affinity Analysis) and load the previously saved .ntp file from the destination folder.
    2. Select the ‘MST’ analysis type.
      Note: If the initial fluorescence in the capillary scan shows signs of ligand-dependent fluorescence effects, it is also possible to choose the ‘initial fluorescence’ analysis type. However, possible ligand-dependent fluorescence effects should be carefully investigated with an SD-test (see Notes).
    3. Add the respective technical or biological repeat runs to the MST analysis by drag-and-drop or by clicking the ‘+’ button below the individual repeat runs.
    4. In order to get information on raw data, MST traces, capillary scans, capillary shape profiles, initial fluorescence, and bleaching rate, click the ‘information’ button below the respective repeat runs.
      Note: It is also possible to inspect these raw data during later steps of the analysis.
    5. Check the automated inspection of the data for sticking effects, as well as aggregation and/or precipitation effects. Also visually inspect the data for these effects (e.g., U-shaped capillary shape peaks, bumps and spikes in the MST traces).
      Note: A detailed description of this quality control step is described in the Notes section and in Figure 2.


      Figure 2. Assay optimization for MST experiments. A.  Example of capillary scan with adsorption/sticking effects in capillaries 1-3 and high-quality samples in capillaries 4-6. B. Example of MST traces showing sample aggregation (left panel) and high quality data (right panel).

    6. Evaluate the data by switching to the dose-response panel.
      Note: Normally, the standard evaluation modes, which automatically adjust the time ranges for determining Fhot and Fcold in order to guarantee an optimal signal-to-noise ratio, are sufficient for most experiments. However, the user is also free to change the evaluation mode to ‘expert’, where it is possible to manually adjust the time ranges used for evaluation of the data.
    7. For determining the KD value of an interaction, select the ‘KD’ model.
      Note: Although the ‘Hill’ model may provide a better fit for the data in many cases, it is advised to choose this model only, when it is justified by known properties of the studied interaction. This could, for example, be a non-1:1 stoichiometry of the interaction or cooperative binding.
    8. For a better comparison of different MST experiments, binding isotherms can be normalized to the fraction of bound molecules (FB) by the following equation:



      where, is the value of an individual MST mix, is the value of the unbound state, i.e., the MST mix with the lowest concentration of the ligand, and is the value of the bound state, i.e., the MST mix with the highest concentration of the ligand.

  2. Example results
    1. Interaction between short repeat peptides of the C-terminal domain of the A. thaliana RNA polymerase II and SPT6L
      1. The synthesis of mRNAs (and other RNAs) by the 12-subunit RNA polymerase II (RNAPII) is of fundamental importance for eukaryotic organisms. Accordingly, transcription by RNAPII is regulated at various steps of the transcription cycle, including polymerase recruitment as well as transcriptional initiation and elongation (Kornberg, 2007). Importantly, the carboxy-terminal domain (CTD) of the largest subunit of RNAPII is differentially modified during the transcription cycle (Buratowski, 2009; Jeronimo et al., 2016). Phosphorylation of the Ser5 residue of the CTD heptapeptide repeats occurs, for instance, during early elongation, while Ser2 is increasingly phosphorylated when elongation proceeds. The differential modification of the RNAPII-CTD is critical for the timely and coordinated recruitment of factors that modulate transcript elongation on chromatin templates as well as of factors that are involved in the processing of mRNAs (5’end capping, splicing, 3’end polyadenylation) (Moore and Proudfoot, 2009; Bentley, 2014). Also in the plant model Arabidopsis thaliana a variety of so-called transcript elongation factors were identified that facilitate RNAPII transcription of repressive chromatin (Van Lijsebettens and Grasser, 2014). One subgroup of these transcript elongation factors is histone chaperones that assist RNAPII by disassembling nucleosomes in the path of the polymerase, thus promoting polymerase progression (Zhou et al., 2015). Examples of these histone chaperones involved in transcriptional elongation are the H2A/H2B chaperone FACT and the H3/H4 chaperone SPT6, both of which are encoded by essential genes in Arabidopsis (Lolas et al., 2010; Gu et al., 2012). SPT6 occurs in Arabidopsis in two variants termed SPT6L and SPT6 (Gu et al., 2012). In a recent study that analyzed the composition of the RNAPII transcript elongation complex by affinity purification from Arabidopsis cells in combination with mass spectrometry, SPT6L–but not SPT6–was identified as a constituent of the complex (Antosz et al., 2017). Since SPT6 from other organisms binds directly to the RNAPII-CTD as studied by fluorescence anisotropy and NMR (Sun et al., 2010; Liu et al., 2011), it was examined whether Arabidopsis SPT6L also interacts with the RNAPII-CTD.
      2. Using MST, binding of the putative SPT6L interaction domain (Phe1218-Asp1412) was analyzed with differentially phosphorylated, synthetic, N-terminally FITC-labelled RNAPII CTD repeat peptides (Antosz et al., 2017). The targets in this experiment were the peptides, which were synthesized with either no phosphorylation, or a phosphoserine at position 2 or 5 and labeled with FITC at their N-terminal residue. The sequences of the peptides were: CTD-noP (N-PSYSPTSPSYSP-C), CTD-Ser2P (N-TSPSY(pS)PTSPSY-C) and CTD-Ser5P (N-SYSPT(pS)PSYSPT-C), with (pS) representing a phosphorylated Ser residue. The ligand in this experiment was the STP6L interaction domain, which was expressed with a GST-tag in E. coli and purified by glutathione-sepharose affinity chromatography (Antosz et al., 2017). The experiments were performed on a Monolith NT.115 device with an MST-power of 40% and an LED-power of 80% at 25 °C.
      3. The MST assay showed that SPT6L(Phe1218-Asp1412) is indeed able to interact with the synthetic CTD peptides and that it has a significantly higher affinity for the peptide with a phosphorylated Ser2 residue. The KD value of this interaction was determined as 134.8 ± 26.6 µM and was highly reproducible over several independent repeats (Figure 3). Experiments with the non-phosphorylated peptide or the Ser5-phosphorylated peptide showed much weaker binding and a reliable determination of the KD values was not possible (estimated KD around 1 mM). It is important to note that this is not an intrinsic issue of the MST technique, but rather a consequence from the concentration of the ligand stock, which was too low to analyze MST mixtures with higher ligand concentrations (highest possible final ligand concentration in an MST mix was 2.15 mM). Thus, the MST assay showed that the interaction domain of SPT6L from A. thaliana also directly binds to the RNAPII-CTD, as has been shown previously for SPT6L proteins from other organisms. Moreover, the assay revealed that the affinity of SPT6L for Ser2-phosphorylated CTD repeat peptides of the RNAPII is at least 10-fold higher than for non-phosphorylated or Ser5-phosphorylated CTDs. These experiments demonstrate how MST can be successfully applied to study interactions between the subunits of sophisticated eukaryotic protein complexes.


        Figure 3. MST analysis of the interactions between GST-SPT6L(Phe1218-Asp1412) and three repeat peptides of the atRNAPII-CTD. The protein SPT6L(Phe1218-Asp1412) served as the unlabelled ligand in this example and was added in increasing concentrations to the three FITC-labelled peptides that served as targets. The peptides resemble specific regions of the C-terminal domain of the A. thaliana RNA polymerase II and were either non-phosphorylated (green), or phosphorylated at Ser2 (red) or Ser5 (blue). Raw fluorescence data were normalized to the fraction of bound target. Error bars represent standard deviations from three individual repeat measurements. The KD was determined from a fit with the ‘KD’ model to the law of mass action (R2 = 0.98). The figure is modified from Antosz et al., 2017 (www.plantcell.org, Copyright American Society of Plant Biologists).

    2. Interactions between histone tail peptides and HP1 proteins
      1. Eukaryotic genomes are usually organized as chromatin, a compact storage form of DNA wound around scaffold proteins. The lowest level of chromatin are nucleosomes, which are octameric complexes of histone proteins circled by a DNA stretch of roughly 150 nucleotides (Kornberg, 1974; Luger et al., 1997). The histone monomers possess flexible, N-terminal tails that are the site of frequent post-translational modifications like acetylations, methylations, or phosphorylations. The frequency and nature of these modifications are important regulators of chromatin structure and DNA accessibility (Jenuwein and Allis, 2001): Modified histone tails can influence interactions between neighboring nucleosomes and can recruit other proteins that influence chromatin structure (Shogren-Knaak et al., 2006). One such ‘reader’ protein is the heterochromatin protein 1 (HP1), a small (around 30 kDa), highly conserved protein that specifically binds to methylated histones, for example to histone tail peptides of histone H3 that feature a bi- or tri-methylation at Lys9 (H3K9met) (Eskeland et al., 2007). Binding of HP1 to methylated histone tails is an important factor in epigenetic regulations and the formation of heterochromatin, i.e., regions of chromatin with low transcriptional activity and high compacting ratio (Kwon and Workman, 2011).
      2. MST enables the researcher to determine the typical interaction parameters of the interactions between HP1 proteins and histone tails in-solution (i.e., without any immobilization) and label-free (i.e., without the need for attaching a fluorescent label to one of the interaction partners). It thus provides the possibility to study these interactions in a system that is very close to the physiological conditions of the interactions. This is possible, because the short histone tail peptides do not contain fluorescent amino acids and at the same time the HP1 proteins we studied contain enough Trp and Tyr residues in order to generate a reliable intrinsic fluorescence signal that can be measured with the Monolith NT.LabelFree MST device. We analyzed the interactions between two HP1 homologs (from Plasmodium falciparum and Drosophila melanogaster) with four different tail peptides of histone H3 (20 amino acids): one unmodified peptide, two di-methylated peptides (at Lys4 and Lys9), and one acetylated peptide (at Lys9).
      3. For each experiment, the HP1 protein served as the target and was used at a final, constant concentration of 3.5 µM. The peptides served as ligands and were assayed at a final concentration, ranging from 10 nm to 10 mM. The Monolith NT.LabelFree was set to an LED power of 20% and an MST power of 40%, with a thermophoresis time interval of 30 sec. Raw fluorescence data were normalized to the fraction of bound ligand and fitted with the ‘KD’ model describing the law of mass action. HP1 from P. falciparum (pfHP1) binds to the di-methylated peptide H3K9(Me2) with a KD value of 4.36 ± 0.14 µM (Figure 4A). Additional experiments showed that this interaction is highly specific, because pfHP1 does not bind to another di-methylated peptide that differs only in the position of the methylated lysine residue (H3K4(Me2)), nor to an acetylated peptide (H3K9(Ac)) or the unmodified peptide (H3) (Figure 4B). We could also show that this interaction specificity is conserved, because also HP1 from D. melanogaster (dmHP1) displays the same binding preferences (Figures 4C and 4D). However, the KD of the interaction with H3K9(Me2) is about 7-fold higher (31.12 ± 0.48 µM).
      4. These experiments also demonstrate that binding isotherms determined with MST are highly reliable. The standard deviations from several individual repeats of a binding experiment are low and the determined KD values are highly reproducible (Figures 4A and 4C). Moreover, the KD value for the interaction between pfHP1 und H3K9(Me2) (4.36 µM) is in good agreement with a value of 7 µM determined by ITC (Jacobs and Khorasanizadeh, 2002). This shows that MST is well suited to study interactions in epigenetic systems. Importantly, the measurements can be done in solution, with very low sample consumption (an important point to consider when working with eukaryotic proteins or synthetic peptides), and also in bioliquids. The technique thus allows for the fast, easy, and flexible characterization of various histone-reader protein interactions.


        Figure 4. MST analysis of the interactions between P. falciparum and D. melanogaster HP1 and different histone peptides using the label-free MST technology. The HP1 proteins from P. falciparum (pfHP1) and D. melanogaster (dmHP1) served as the targets in this example. In this example, it was not necessary to attach a fluorescent label to the targets, because both proteins featured high enough molar extinction coefficients to analyze the interactions on a Monolith NT.LabelFree device. A prerequisite for this was also that the histone peptides did not contain tryptophan and tyrosine residues (no fluorescence above 300 nm).

Notes

  1. Detection of adsorption and capillary sticking effects and troubleshooting
    The capillary scan can detect sticking or adsorption effects that occur between the targets and the glass surface of the capillaries. Irregular peak shapes (Figure 2A, capillaries 1-3), such as U-shaped or flattened peaks, indicate such sticking or adsorption effects. In many cases a change of capillary type (standard treated, premium coated, or hydrophobic) can prevent them, resulting in regularly shaped peaks (Figure 2A, capillaries 4-6). If a change of capillary type does not enhance the capillary scan peak shapes, a change in buffer conditions or the addition of detergents like Tween (0.005-0.1%) or Pluronic F-127 (0.01-0.1%) may prevent sticking and adsorption. Generally, a capillary test should be performed with one MST mix prior to the measurement of the full 16 MST mixes in order to find conditions where no sticking and adsorption occurs. This stage of an MST experiment is essential for high data quality later on.
  2. Detection of fluorescence effects and troubleshooting
    1. The initial capillary scan also provides information on possible pipetting errors or a ligand-dependent change in fluorescence emission. Pipetting errors are reflected in random height differences of the capillary peaks. A high pipetting accuracy is mandatory to avoid such errors. Also, buffer dilution effects should be avoided, i.e., the buffer used for preparing the dilution steps should be exactly the same buffer as the one of the ligand working solution. Large differences in the fluorescence readout between individual capillaries may also be a sign of sample aggregation. Aggregates are characterized by a much higher fluorescence density and thus increase the fluorescence readout when coming into the optical readout focus.
    2. Systematic changes in the fluorescence signal that correlate with the ligand concentration, however, hint at a possible fluorescence enhancement or quenching effect by the ligand. In order to exclude other possibilities, like unspecific fluorescence decrease, for these systematic changes, an SD-test is carried out: If the systematic change of fluorescence is really ligand-dependent, this effect should not be detectable under denaturing conditions and the fluorescence signal should become identical for all capillaries. If, however, the fluorescence signal still shows deviations under denaturing conditions, the reason is probably a loss of fluorescently labeled molecule due to adsorption, sticking, or aggregation effects. A standard SD-test is performed as follows: 10 µl of the first MST mix and 10 µl of the last MST mix are each transferred to a fresh PCR tube containing 10 µl of a 2x SD mix (4% SDS, 40 mM DTT). After mixing, the samples are incubated at 95 °C for 5 min to ensure denaturation. Tow fresh capillaries are filled and the fluorescence intensities are recorded at the same settings as before.
  3. Detection of aggregation effects and troubleshooting
    Sample aggregation and/or precipitation can easily be detected in the MST traces as bumps and spikes (Figure 2B, left panel). Samples with no aggregation usually display smooth MST traces (Figure 2B, right panel). If aggregation occurs in a sample, a change of capillary type, buffer conditions and additives such as detergents (0.005-0.1% Tween-20, 0.01-0.1% Pluronic F-127, or similar) or BSA (> 0.5 mg/ml), pH conditions, and salt concentrations may optimize solubility of the molecules. Large aggregates may be removed prior to the measurement by centrifugation (at least 10 min at 14,000 x g). In order to ensure optimal data quality, the MST traces should resemble the ones shown in the right panel of Figure 2B.
  4. Data analysis and curve fitting
    1. In principle, MST traces can be evaluated with respect to the different underlying physical phenomena. The temperature jump (or T-jump) describes the sudden change if fluorescence emission upon temperature change, which is often characteristic for different fluorophores. The T-jump is highly affected by binding of the ligand in close proximity to the fluorophore. The slower thermophoresis phase of an MST trace is influenced by the movement of the molecules along the induced temperature gradient and is thus highly sensitive to changes in size, charge, and hydration shell of the molecules under study.
    2. In the ‘expert’ analysis mode of the MO.Affinity Analysis software the user can vary the time points chosen for either T-jump or thermophoresis evaluation. If aggregation occurs in the samples and the instructions described above do not resolve these issues, it may be possible to use the early parts of the thermophoresis phase where aggregation effects may be smaller than in later parts. Examining the early parts of the thermophoresis phase may also increase data quality due to less-pronounced convection effects inside the capillaries.
    3. The MO.Affinity Analysis software offers two options for fitting the dose-response curves. The ‘KD’ model is based on the law of mass-action:



      where, T is the labeled target and L is the non-fluorescent ligand.
      The fluorescence signal that is dependent on the concentration of the titrated ligand (F(c𝐿)) can be calculated as follows:



      where, Fu and Fb are the fluorescence values in the unbound and bound states, and cTL and cT are the concentrations of formed complex and the fixed concentration of the fluorescently labelled target.
      From the fraction bound value , the KD of an interaction can be derived:



      where, KD is the dissociation constant.
      Note that this fitting model is only valid for data that describe a 1:1 interaction between the target T and the ligand L with one specific binding site or multiple binding sites with the same affinity. The ‘Hill’ model allows for determining the EC50 value of an interaction:



      In this case, the fraction bound is given as:




      where, cL is the concentration of the titrated, non-labelled ligand.
      Note that the ‘Hill’ model is often used for interactions with cooperative binding. It should not be used for fitting data that can be clearly explained with the law of mass action. Also, the ‘Hill’ model only determines the EC50 value of a molecular interaction, which is the concentration of the ligand where 50% of the target molecules are bound. It is important to know that the EC50 value does not directly correspond to the dissociation constant KD. It is more an apparent measure of affinity for one particular experiment that is dependent on the used concentrations and conditions.
  5. Detection of low fluorophore concentration
    Concentrations of labeled targets below 1 nM usually require high excitation light intensities (LED power > 75% (or ‘high’) on the Monolith NT.115pico). Such high intensities can lead to significant photobleaching of the fluorophore and thus introduce additional noise to the data. NanoTemper Technologies offers an anti-photobleaching kit that can help reduce these effects.
  6. About the temperature gradient
    The temperature gradient spans 2 °C at an MST power of 20% and 6 °C at an MST power of 80%. The total volume of the heated sample is 2 nl. It is recommended to identify the optimal MST power prior the full binding experiment by performing several measurements with a single capillary and different MST power settings (for example 20%, 40%, and 80%).

Recipes

  1. NaP buffer
    10 mM sodium phosphate pH 7.0
    1 mM EDTA
    1 mM DTT
    0.5 mM PMSF
  2. MST-T buffer
    50 mM Tris-HCl pH 7.8
    150 mM NaCl
    10 mM MgCl2
    0.05% Tween-20

Acknowledgments

We thank Corinna Kuttenberger and Clemens Entzian for expert scientific and technical assistance.

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简介

蛋白质 - 肽相互作用是许多生理过程的一部分,例如表观遗传学,其中组蛋白复合物的肽区域对于染色质结构的调节是至关重要的。短肽通常也被用作小分子药物靶向蛋白质复合物的替代物。研究蛋白质和肽之间的相互作用是系统生物学,细胞生物学,生物化学和药物设计中的重要任务。然而,这一任务往往受到经典生物物理学方法分析分子间相互作用的缺陷的困扰,例如表面等离子体共振(SPR)或等温滴定量热法(ITC),其需要固定相互作用配偶体或非常高的样品浓度。 MicroScale热泳(MST)是一种创新的方法,可以确定分子间相互作用的重要参数,如解离常数,化学计量和热力学。而且,它可以快速准确地进行,可以自由选择缓冲液或生物液体,不需要固定样品,样品消耗也非常少。这里我们详细描述了两个MST测定法,其分析(i)真核RNA聚合酶II的某些肽段与真核转录延伸复合物的蛋白质亚基之间的相互作用和(ii)N-末端组蛋白尾肽与表观遗传学之间的相互作用读者蛋白质。这些实验表明,MST能够表征蛋白质 - 肽相互作用,这些相互作用仅由肽的微小变化触发,例如,在特定的丝氨酸残基处仅有一个磷酸化。

【背景】生物学背景:蛋白质 - 蛋白质相互作用(PPIs)对于几乎所有的细胞过程都是至关重要的,包括DNA复制,转录和翻译,包括代谢酶复合物和大型细胞机制的形成,生物信号的传递。考虑到PPI的生理重要性,蛋白质复合物被认为是药物开发中的高价值目标(Milroy等人,2014; Nevola和Giralt,2015)。然而,传统的基于小分子的靶向方法常常不能产生有效的抑制剂,这主要是由于大的且通常是扁平的蛋白质界面(Sperandio等人,2010)。替代策略涉及通常模拟相互作用伙伴的结合表面的一部分的基于肽的抑制剂(Boersma等人,2012; Azzarito等人,2013; Arkin等人,等,2014; Pelay-Gimeno等,,2015)。蛋白质和肽之间的典型5-20个氨基酸之间的这种相互作用常常在自然界中发现,例如抗体,激酶,磷酸酶,MHC蛋白质或表观遗传读取器蛋白质(Stanfield and Wilson,1995)。此外,用小肽特异性靶向PPIs可以帮助阐明大型复杂蛋白质复合物的组成和功能,如真核生物RNA聚合酶II延长复合物(Antosz等人,2017)或特异性表观遗传读取器蛋白(Josling等人,2015)。

技术背景为了精确和全面地表征蛋白质 - 蛋白质和蛋白质 - 肽在亲和力,动力学和热力学方面的相互作用,已经开发了一系列生物物理技术和方法。公认的技术是等温滴定量热法(ITC)(Pierce等人,1999; Ghai等人,2012),基于表面等离子体共振(SPR)的测定法Pattnaik,2005; Ishii等人,2013),天然质谱(Heck,2008; Yan等人,2017),生物层干涉测量法(Abdiche等, 2008年;康塞普西翁等人,2009年),静态或动态光散射(Wen等人,1996; Kameyama和Minton,2006; Hanlon等人,2010),以及荧光光谱学(Lundblad等人,1996; Lin等人,2014; Raines等人, ,2015)。虽然这些方法中有一些独特地提供了特定的信息,例如上下速率或精确的复杂化学计量,但是它们通常是实验室强烈的和复杂的,由于高样品消耗,低灵敏度,表面固定化,传质限制,以及缓冲限制或不能量化涉及小肽的相互作用。创新的MicroScale热泳技术(MST)是一种功能强大的技术,可以克服这些局限性,可以以非常低的样品消耗快速,精确,经济高效地控制分子间相互作用,无需固定样品,自由选择缓冲液或bioliquids。 MST使研究人员能够确定分子相互作用的重要参数,例如结合亲和力(来自皮摩尔摩尔摩尔),结合化学计量学和相互作用热力学(Wienken等人,2010; Jerabek- Willemsen等人,2011年)。重要的是,MST对相互作用分子的分子量没有任何限制,因此量化几百kDa大小的蛋白质与分子量低于1kDa的短肽之间的相互作用是直接的和精确的(Antosz等人, 2017)。
热泳的物理现象:“热泳”的物理现象描述了分子沿温度梯度的运动。这种运动取决于分子的大小,电荷和水合壳(Braun and Libchaber,2002; Duhr and Braun,2006)。蛋白质和配体(其他蛋白质,肽,DNA,RNA,小分子)之间的相互作用改变这些参数中的至少一个,导致沿着温度梯度的不同的热泳迁移率(Jerabek-Willemsen等人,2014)。因此,大蛋白质和小配体(如短肽)之间的相互作用只会导致尺寸和电荷的微小变化,这将显着影响由于水合壳变化引起的热泳。

MicroScale Thermophoresis MicroScale Thermophoresis是利用热泳原理研究溶液中分子间相互作用的创新技术(Duhr等人,2004; Baaske等人, 2010年; Jerabek-Willemsen等人,2011年)。通过使用红外(IR)激光器在非常薄的玻璃毛细管内产生微观温度梯度来诱导分子的热泳运动。在图1A中示出了MST装置(Monolith NT.115,德国慕尼黑的NanoTemper Technologies GmbH)的相应技术设置。该梯度集中在约50μM的直径,并且包括温度差ΔT为2-6℃。在相同的区域中,通过使用蛋白质或肽中色氨酸的固有荧光(Seidel等人,2012)或荧光信号的荧光信号追踪分子的热泳运动(Schubert等人,2012; Zillner等人,2012)中的一种相互作用。在加热时,分子在温度梯度的中心消耗或积累,并且荧光光学元件可以通过Soret系数来量化,其中使用表示毛细管的加热和未加热区域中的浓度,ΔT表示沿着毛细管的温度差梯度(Duhr和Braun,2006):



图1B描绘了一个典型的MST测量。一开始,在固定的起始温度下测量毛细管中的荧光通常为5秒。然后由IR激光器引起温度梯度,导致测量的荧光信号急剧下降,即所谓的温度或T跳跃。该信号跳跃是由于使用的荧光团的量子产率随温度而变化的结果。之后,对应于Soret系数的光学聚焦中的荧光分子的较慢的热泳导致的耗尽导致荧光信号的指数下降。在通常20-30秒的测量时间之后,再次关闭IR激光器,这导致荧光分子的热泳反向扩散进入光学焦点并伴随着相反的T跳跃。

为了确定所研究的复合物的平衡解离常数(K D),使用固定量的荧光结合配偶体和增加量的非定量的MST测量来进行一系列MST测量荧光伴侣被记录(图1C)。 16个MST迹线相对于其初始荧光被归一化。非荧光伙伴的浓度范围选择的方式是,最低浓度导致实际上完全“未结合”的荧光伙伴,最高浓度导致完全“结合”的荧光伙伴。通常将非荧光配偶体的16倍稀释物制备成在投射的KD低于约10倍至10倍的范围内,补充相同量的荧光合作伙伴并装入16个人毛细血管。通常将荧光配偶体的浓度保持在低于预计的K d,以允许精确确定真实的平衡亲和力。然后使用16个MST迹线中的每一个的“冷”和“热”状态之间的差异来确定每条迹线的荧光变化:



相对于配体浓度绘制这些值最终导致典型的结合等温线,其产生相互作用的K D值(图1D)。

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图1. MicroScale热泳。A. MST设备的技术设置。分子在玻璃毛细管内部的热泳运动由聚焦在50μM宽区域上的红外激光引起。在相同的区域使用荧光发射来跟踪这个运动。总共可以在一个装置中随后分析16个毛细管。 B.一个MST跟踪的例子。在五秒钟的初始延迟之后,打开IR激光器以建立温度梯度。在T跳跃阶段之后,热泳运动导致光学焦点中的荧光分子呈指数下降。通常20-30秒的测量时间之后,关闭激光器。 C.一个典型的MST实验的结合痕迹,其中16个毛细管被分析,其含有相同浓度的荧光相互作用配偶体,但增加了非荧光配偶体的浓度。所有的痕迹归一化为1的相同初始相对荧光值。D.通过绘制标准化荧光与非荧光结合配偶体(配体)浓度的差异得到的最终结合等温线。图由Entzian和Schubert修改,2016;最初由慕尼黑的NanoTemper Technologies提供。

MST的优点和缺点:与其他生物物理方法相比,MST提供了几个整体优势来表征蛋白质 - 蛋白质和蛋白质 - 肽相互作用。首先,填充毛细管的MST痕迹和横向扫描提供了直接的样品质量控制,因为它们允许容易地检测聚集,沉淀和吸附效应。这使研究人员能够快速改变和优化技术和缓冲条件,以提高样品的稳定性和数据质量。重要的是,这16个毛细血管的控制时间不到两分钟。此外,获得均衡所需的全部测量值15分钟15分钟。此外,MST是无固定的,因此可以确定几乎所有缓冲液中的亲和力,甚至在复杂的生物液体如裂解物和血清中也具有亲和性(Wienken等人,2010; Seidel等人, ,2013)。而且,MST的样品消耗量非常低,在与难生产的真核生物蛋白质一起工作时,这可能是一个至关重要的问题。通常,荧光相互作用配偶体以非常低的纳摩尔浓度(5-20nM)进行测定,而非荧光配偶体通常 - 取决于纳摩尔浓度的KD-到低微摩尔范围。最后,可检测亲和力(pM至mM)的高动态范围和不存在的大小限制允许表征涉及蛋白质,肽,小分子,DNA和RNA的各种不同的分子相互作用,仅举几例。但是,MST不允许确定分子相互作用的开关速率。尽管如此,从MST实验得到的结合参数与已建立的最先进的方法如SPR或ITC(Ramakrishnan等人,2012; Chen等人, ,2015; Stoltenburg等人,2015; Wan等人,2015; Harazi等人,2017)。最后,必须指出的是,对于大多数MST应用,一个交互伙伴必须用荧光团进行修饰。因此,如果肽或蛋白质都不能用荧光团标记(例如,因为两者都不含荧光团偶联的赖氨酸或半胱氨酸残基),所以不能使用标准的MST。然而,在这种情况下,由于读出蛋白质或肽的内在酪氨酸和色氨酸荧光,无标记MST分析可以是另一种选择。

关键字:微量热泳, 分子相互作用, 结合亲和力, 结合参数, 蛋白质 - 肽相互作用, 组蛋白, 表观遗传学, RNA聚合酶

材料和试剂

  1. 低体积低结合反应管(例如,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:90410)
  2. Eppendorf管(1.5ml)(Sigma-Aldrich,目录号:T9661) 制造商:Eppendorf,目录号:022363204。
  3. PCR管(200μl,NanoTemper Technologies GmbH,Munich,Germany)
  4. 移液器吸头(10μl,100μl,1,000μl)(STARLAB INTERNATIONAL,目录号:S1111)
  5. 拟南芥RNA聚合酶II(Biomatik,Wilmington,Delaware,USA)CTD的FITC标记的肽
    注:“pS”表示磷酸化的丝氨酸;在N-末端用异硫氰酸荧光素[FITC,CAS号:27072-45-3]标记肽。
    1. CTD-noP(序列:PSYSPTSPSYSP)
    2. CTD-Ser2P(序列:TSPSY(pS)PTSPSY)
    3. CTD-Ser5P(序列:SYSPT(pS)PSYSPT)
  6. 蛋白质SPT6L(Phe1218-Asp1412)
    注意:如(Kammel et al。,2013)所述,在具有GST标签的大肠杆菌中表达,并通过谷胱甘肽 - 琼脂糖亲和层析纯化。
  7. 来自恶性疟原虫的蛋白质HP1( P.falciparum )
    注:在具有六聚组氨酸标签的大肠杆菌BL21-CodonPlus(DE3)-RIL细胞中表达并通过Ni2 + -IMAC层析(Josling等,2015)纯化。 em>
  8. 来自果蝇( D. melanogaster )的蛋白HP1
    注:在具有六聚组氨酸标签的大肠杆菌BL21-CodonPlus(DE3)-RIL细胞中表达并通过Ni纯化 2 + -IMAC色谱法(Josling等,2015)。
  9. 组蛋白肽
    所有组蛋白肽均购自ANASPEC,Kaneka Eurogentec(比利时列日)
  10. Pluronic F-127(CAS编号:9003-11-6)(Sigma-Aldrich,目录号:P2443)
  11. 十二烷基硫酸钠钠盐(SDS; CAS号:151-21-3)(Sigma-Aldrich,目录号:L3771)
  12. 磷酸钠(CAS编号:7601-54-9)(Sigma-Aldrich,目录号:342483)
  13. 乙二胺四乙酸(EDTA,CAS编号:60-00-4)(Sigma-Aldrich,目录号:E9884)
  14. DL-二硫苏糖醇(DTT,CAS号:3483-12-3)(Sigma-Aldrich,目录号:43815)
  15. 苯基甲磺酰氟(PMSF,CAS号:329-98-6)(Sigma-Aldrich,目录号:78830)
  16. 三(羟甲基)氨基甲烷(Tris,CAS号:77-86-1)(Sigma-Aldrich,目录号:T1503)
  17. 氯化钠(NaCl,CAS编号:7647-14-5)(Sigma-Aldrich,目录号:S7653)
  18. 氯化镁(MgCl 2,CAS号:7786-30-3)(Sigma-Aldrich,目录号:M8266)
  19. 吐温20(CAS编号:9005-64-5)(Sigma-Aldrich,目录号:P1379)
  20. NaP缓冲液(见食谱)
  21. MST-T缓冲液(见食谱)

设备

  1. 移液器(Eppendorf Reference 20,100,200,1000)(Eppendorf,产品目录号:4920000032,4920000059,4920000067,4920000083)
  2. MiscroScale热电泳仪Monolith NT.115(NanoTemper Technologies,型号:Monolith NT.115)
  3. MiscroScale热电泳仪Monolith NT.LabelFree(NanoTemper Technologies,型号:Monolith NT.LabelFree)
  4. Monolith NT TM毛细管“高级涂层”(NanoTemper Technologies,慕尼黑,德国)
  5. 培养箱(Eppendorf,型号:ThermoMixer®舒适型,目录号:5360000011)
  6. 离心机(Eppendorf,型号:5424R,目录号:5404000219)

软件

  1. MO.Control(NanoTemper Technologies,德国慕尼黑)
  2. MO.Affinity分析(NanoTemper Technologies,德国慕尼黑)

程序

  1. 解决方案准备
    注:缓冲区
    1. 对于SPT6L(Phe1218-Asp1412)和拟南芥RNA聚合酶II的CTD肽之间的相互作用,测定缓冲液是NaP缓冲液(参见食谱)。
    2. 对于HP1蛋白与组蛋白肽之间的相互作用,测定缓冲液是MST-T缓冲液(见食谱)。

    1. 准备工作的解决方案的目标
      1. 肽CTD-noP,CDT-Ser2P和CDT-Ser5P:这些肽用作SPT6L(Phe1218-Asp1412)结合的靶标。
        1. 按照制造商的说明,溶解肽。

        2. 用MST-T缓冲液稀释肽溶液至80 nM,制备目标工作溶液
      2. 来自 P的HP1蛋白。恶性肿瘤和 D。果蝇。这些蛋白质作为不同组蛋白肽结合的靶标。靶标的标记在这种情况下是不必要的,因为它们固有的色氨酸荧光可以与Monolith NT.LabelFree设备一起使用。
        通过在MST-T缓冲液中稀释蛋白质储备液至3.5μM来准备目标工作溶液。
    2. 制备配体的工作溶液
      1. SPT6L(Phe1218-Asp1412)蛋白质:该蛋白质用作CTD-肽结合的配体。
        1. 用MST-T缓冲液稀释配体原液至4.3mM。理想情况下,配体的工作溶液浓度应比预计的K / D值高约50倍。
          注意:在4℃下以14,000×g离心配体原液5分钟可能有助于除去聚集体。建议使用低容量低结合反应管(如Thermo Fisher Scientific),以避免分子吸附到管壁上。
        2. 当制备配体工作溶液时,必须考虑到(取决于稀释因子),工作溶液中仍然存在一定量的配体原液缓冲液。如果超过1%,我们建议相应地调整最终的MST缓冲区。
      2. 组蛋白肽:这些肽充当用两种HP1蛋白质靶标进行测量的配体。
        1. 按照制造商的说明,溶解肽。

        2. 在NaP缓冲液中稀释肽溶液至20 mM,制备配体工作原液
    3. 配体稀释系列的制备
      1. 配体在200μlPCR管中以16个连续步骤稀释。
        在16个步骤的每个步骤中稀释配体50%(1:1稀释)。
        注意:在控制软件中实现的浓度查找工具可模拟结合数据,并有助于找到稀释系列的正确浓度范围。

      2. 添加10μL检测缓冲液到PCR管2至16
      3. 在PCR管1中填充20μl配体工作溶液。
      4. 将PCR管1中的10μl转移到PCR管2中,并通过上下移液数次来正确混合。
        注意:避免涡流或剧烈摇动,以防止蛋白质变性。
      5. 从PCR管2中将10μl转移到PCR管3中并混合。对剩余的稀释步骤重复此过程。
      6. 丢弃从最后一个管多余的10微升。
        所有16个PCR管应该含有10μl的体积 注意:避免任何缓冲液稀释效应是很重要的。所有16个PCR管应含有完全相同的缓冲液组成。如果配体储备溶液与MST测定缓冲液处于不同的缓冲液中,则最佳实践是制备MST测定缓冲液的等分试样,其具有与配体工作溶液的缓冲液完全相同的组成。这个缓冲液然后用于移液连续稀释系列。例如,如果配体原液在缓冲液A + 2mM DTT中并在缓冲液A中稀释10倍以产生配体工作溶液,则工作溶液中的DTT浓度为0.2mM。因此,使用缓冲液A + 0.2mM DTT来制备系列稀释系列。
    4. 制备最终的MST混合物
      1. 尽管4μl的体积足以填充MST毛细血管,但建议准备至少20μl的最终MST混合物,以尽量减少移液错误。
      2. 向每个10μl配体稀释步骤中加入10μl目标工作溶液,并通过上下吸移几次来正确混合。
        注意:避免涡流或剧烈摇动,以防止蛋白质变性。
      3. 在室温下孵育五分钟以达到结合平衡。取决于具体的靶标和配体,可能需要更长的温育时间或不同的温育温度。具体的孵育时间和温度必须根据关于要分析的相互作用的先验知识来选择。作为一般建议,我们建议在室温下培养5到20分钟。
      4. 通过将毛细管浸入样品中,用16个MST混合物填充16个毛细管。
        注意:请勿触摸将要进行光学测量的中间部分的毛细管。
      5. 将毛细管放在毛细管托盘上,启动MST设备。

  2. MST测量
    1. 启动MST设备
      1. 启动MST控制软件并通过启用温度控制来调节所需的温度。通常在25°C下进行测量。等待温度达到预定值。
        注意:MST仪器可以在22至45°C的温度范围内进行温度控制。
      2. 将毛细管托盘放入MST设备。
      3. 将FITC荧光标记的LED通道(荧光激发)设置为“蓝色”,并将LED电源设置为在具有标准传感器的MST设备处获得300至1,000个单位的荧光信号,在具有高分辨率的设备处获得6,000至18,000个荧光信号,灵敏度传感器。
        注:其他荧光标签可能需要“红色”LED设置。检查使用的荧光团的激发波长。
    2. 毛细管扫描
      1. 执行毛细管扫描,以检查样品的不同质量方面。
      2. 检查毛细管扫描,看是否获得最大荧光信号在上述范围内。
      3. 检查毛细管扫描是否有标记的靶向毛细血管玻璃壁的粘连效应。这导致U形或平坦的山峰。
      4. 检查毛细管扫描的移液错误。这导致了16个MST混合物的荧光值不一致。
      5. 检查配体依赖性荧光增强或淬灭效应的毛细管扫描。这些导致随着16 MST混合物中配体浓度的增加而增加或减少荧光值。
    3. MST测量
      1. 在开始MST测量之前,确保排除任何粘着效应,移液错误,增强/猝灭效应,并确保足够的荧光信号。
      2. 在对照软件中将16个MST混合物中的每一个与相应的最终配体浓度分配。在较新的控制软件版本中,只设定最高的配体浓度(通常在MST混合物1中),并选择稀释类型(例如1:1或1:2)。
        注意:此时必须输入的配体浓度是上述配体稀释系列浓度的一半,因为稀释样品与目标溶液1:1混合。
      3. 输入荧光标记目标的固定浓度。
        注意:由于上述MST混合物的制备,该浓度也是目标工作溶液浓度的一半。
      4. 对于大多数应用,默认设置(5秒的初始荧光,30秒的热泳记录和5秒的热泳后荧光)就足够了。
      5. 调整MST功率为20%。
        注意:
        1. 为了获得最佳的信噪比并避免非特异性效应,建议使用20-40%的激光功率。在某些情况下,可能需要更高的激光功率来获得未结合和结合分子的良好分离。
        2. 在较新的控制软件版本中,只有MST电源设置“低”,“中”和“高”可用。对于大多数应用程序,建议使用“中”。
      6. 输入将保存实验文件的目标文件夹路径。实验将被保存为.ntp文件。
      7. 开始MST测量。
        。根据设定的时间,测量需要10-15分钟。
      8. 重复MST测量至少两次,以更可靠地确定平衡结合亲和力。
        注意:为了测试测量的技术可重复性,相同的毛细管可以用于多个MST测量。

数据分析

  1. 数据分析
    1. 启动MST分析软件(MO.Affinity Analysis),并从目标文件夹中加载以前保存的.ntp文件。
    2. 选择“MST”分析类型。
      注意:如果毛细管扫描中的初始荧光显示配体依赖性荧光效应的迹象,则也可以选择“初始荧光”分析类型。然而,应该用SD测试仔细研究可能的配体依赖性荧光效应(参见注释)。
    3. 通过拖放或单击重复运行下方的“+”按钮,将相应的技术或生物重复运行添加到MST分析。
    4. 为了获得有关原始数据,MST曲线,毛细管扫描,毛细管形状曲线,初始荧光和漂白率的信息,请单击相应重复运行下方的“信息”按钮。
      注意:也可以在分析的后续步骤中检查这些原始数据。
    5. 检查粘附效应数据的自动检查,以及聚集和/或降水效应。同时目测检查这些效应的数据(例如,U形毛细管形状峰,MST迹线中的凸起和尖峰)。
      注意:这个质量控制步骤的详细描述在注释部分和图2中有描述。


      图2. MST实验的测定优化A.毛细管扫描吸附/粘附效应的实例1-3毛细管中的高质量样品4-6。 B.示例MST跟踪显示样本聚合(左图)和高质量数据(右图)。

    6. 通过切换到剂量反应面板来评估数据。
      注:正常情况下,标准的评估模式,自动调整确定Fhot和Fcold的时间范围,以保证最佳的信噪比,对于大多数实验来说是足够的。然而,用户也可以自由地将评估模式改变为“专家”,其中可以手动调整用于评估数据的时间范围。
    7. 要确定交互的 K D 值,请选择 K / em> '模型。
      注意:虽然'希尔'模型在很多情况下可以更好地适用于数据,但是当被研究的相互作用的已知属性证明时,建议只选择这个模型。例如,这可能是相互作用或合作结合的化学计量比 -1:1。
    8. 为了更好地比较不同的MST实验,可以通过以下等式将结合等温线归一化为结合分子(FB)的分数:



      其中,是单个MST混合的值,是未绑定状态的值, MST混合物具有最低浓度的配体,并且<! - SIPO

  2. 示例结果
    1. 拟南芥RNA聚合酶II的C-末端结构域的短重复肽与SPT6L之间的相互作用
      1. 通过12亚基RNA聚合酶II(RNAPII)合成mRNA(和其他RNA)对于真核生物是非常重要的。因此,RNAPII的转录在转录周期的各个步骤中被调节,包括聚合酶募集以及转录起始和延伸(Kornberg,2007)。重要的是,RNAPII的最大亚基的羧基末端结构域(CTD)在转录周期中被不同地修饰(Buratowski,2009; Jeronimo等人,2016)。 CTD七肽重复的Ser5残基的磷酸化例如在早期延伸期间发生,而当延伸进行时Ser2越来越磷酸化。 RNAPII-CTD的差异修饰对调控染色质模板上的转录延长的因子及与mRNA加工相关的因子(5'端加帽,剪接,3'末端多聚腺苷酸化)(Moore和Proudfoot,2009; Bentley,2014)。同样在拟南芥植物模型中,鉴定了促进RNA印迹抑制染色质RNA转录的多种所谓的转录延伸因子(Van Lijsebettens and Grasser,2014)。这些转录本延长因子的一个亚组是通过在聚合酶途径中分解核小体来辅助RNAPII从而促进聚合酶进展的组蛋白伴侣(Zhou等人,2015)。涉及转录延伸的这些组蛋白伴侣的实例是H2A / H2B伴侣FACT和H3 / H4伴侣SPT6,两者都是由拟南芥中的必需基因编码的(Lolas等人, 2010年; Gu等人,2012年)。 SPT6以称为SPT6L和SPT6的两种变体形式存在于拟南芥中(Gu等人,2012)。在最近的研究中,通过结合质谱分析从拟南芥细胞中亲和纯化来分析RNAPII转录物延长复合物的组成,发现SPT6L(而不是SPT6)是复合物的组成成分(Antosz <等),2017)。由于通过荧光各向异性和NMR研究,由于来自其他生物体的SPT6直接结合到RNAPII-CTD(Sun等人,2010; Liu等人,2011),所以它检查了拟南芥属SPT6L是否也与RNAPII-CTD相互作用。
      2. 使用MST,用差异磷酸化的,合成的,N-末端FITC标记的RNAPII CTD重复肽(Antosz等人,2017)分析推定的SPT6L相互作用结构域(Phe1218-Asp1412)的结合。这个实验中的目标是这样的肽,其不用磷酸化合成,或者在2或5位被磷酸丝氨酸合成,并在其N端残基上用FITC标记。 (序列为:CTD-noP(N-PSYSPTSPSYSP-C),CTD-Ser2P(N-TSPSY(pS)PTSPSY-C)和CTD-Ser5P(N-SYSPT(pS)PSYSPT-C) pS)代表磷酸化的Ser残基。该实验中的配体是STP6L相互作用域,其在E中用GST标签表达。并通过谷胱甘肽 - 琼脂糖亲和层析法纯化(Antosz等人,2017)。实验在Monolith NT.115器件上进行,MST功率为40%,LED功率为80%,温度为25°C。
      3. MST测定显示SPT6L(Phe1218-Asp1412)确实能够与合成的CTD肽相互作用,并且对于具有磷酸化Ser2残基的肽具有显着更高的亲和力。该相互作用的K d值被确定为134.8±26.6μM,并且在几个独立的重复中高度重现(图3)。用非磷酸化肽或Ser5-磷酸化肽进行的实验显示出弱得多的结合,并且不可能确定K + D值是不可能的(估计在约1mM的K D )。重要的是要注意,这不是MST技术的固有问题,而是来自配体原料浓度的结果,其配体原料的浓度太低,不能分析具有更高配体浓度的MST混合物(MST中最高可能的最终配体浓度混合物是2.15mM)。因此,MST测定显示来自拟南芥的SPT6L的相互作用结构域也直接结合于RNAPII-CTD,如之前已经显示的来自其他生物体的SPT6L蛋白质。此外,测定显示SPT6L对于RNAPII的Ser2-磷酸化CTD重复肽的亲和力比对于非磷酸化或Ser5-磷酸化CTD至少高10倍。这些实验展示了MST如何成功应用于研究复杂的真核蛋白复合物亚基之间的相互作用。


        图3.GST-SPT6L(Phe1218-Asp1412)与atRNAPII-CTD的三个重复肽之间的相互作用的MST分析。在该实施例中,蛋白质SPT6L(Phe1218-Asp1412)充当未标记的配体,并且以递增浓度添加到用作靶的三种FITC标记的肽中。这些肽类似于拟南芥RNA聚合酶II的C-末端结构域的特定区域,并且在Ser2(红色)或Ser5(蓝色)上是非磷酸化(绿色)或磷酸化的。将原始荧光数据标准化为结合靶标的分数。误差线表示三次重复测量的标准偏差。根据质量作用的规律,从与模型的拟合中确定了K R 2 = 0.98)。该图从Antosz et。,2017( www.plantcell.org ,美国植物生物学家协会版权)。

    2. 组蛋白尾肽与HP1蛋白之间的相互作用
      1. 真核生物基因组通常被组织成染色质,一种围绕脚手架蛋白质的紧密的DNA存储形式。染色质的最低水平是核小体,它是由约150个核苷酸的DNA片段(Kornberg,1974; Luger等,1997)环绕的组蛋白的八聚体复合物。组蛋白单体具有灵活的N-末端尾部,这是经常发生翻译后修饰的位点,如乙酰化,甲基化或磷酸化。这些修饰的频率和性质是染色质结构和DNA可及性的重要调节因子(Jenuwein和Allis,2001):修饰的组蛋白尾巴可以影响相邻核小体之间的相互作用并且可以招募其他影响染色质结构的蛋白质(Shogren-Knaak等人,2006)。一种这样的“读取器”蛋白是异染色质蛋白1(HP1),一种小的(约30kDa)高度保守的蛋白质,其特异性结合甲基化的组蛋白,例如组蛋白H3的组蛋白尾肽,在Lys9(H3K9met)处的甲基化(Eskeland等人,2007)。 HP1与甲基化组蛋白尾部的结合是表观遗传学调控中的一个重要因素,也是形成异染色质(即染色质区域)的低转录活性和高致密比(Kwon and Workman,2011)。 />
      2. MST使得研究人员能够确定HP1蛋白和组蛋白尾部在溶液中(而不是任何固定化)和无标记( ie )之间的相互作用的典型相互作用参数,而不需要将荧光标签附加到交互伙伴之一)。因此,它提供了在与相互作用的生理条件非常接近的系统中研究这些相互作用的可能性。这是可能的,因为短的组蛋白尾肽不含有荧光氨基酸,同时我们研究的HP1蛋白含有足够的Trp和Tyr残基,以产生可用Monolith NT测量的可靠的内在荧光信号。 LabelFree MST设备。我们用四种不同的组蛋白H3(20个氨基酸)的尾肽分析了两种HP1同系物(来自恶性疟原虫和黑腹果蝇)之间的相互作用:一种未修饰的肽, (在Lys4和Lys9处)和一个乙酰化肽(在Lys9处)。
      3. 对于每个实验,HP1蛋白作为靶标,并以3.5μM的最终恒定浓度使用。该肽用作配体,并以10nm至10mM的最终浓度进行测定。 Monolith NT.LabelFree被设置为20%的LED功率和40%的MST功率,具有30秒的热泳时间间隔。将原始荧光数据标准化为结合配体的分数,并用描述质量作用定律的“KD”模型拟合。来自 P的HP1。恶性疟原虫(pfHP1)结合具有4.36±0.14的KD值的二甲基化肽H3K9(Me2) μM(图4A)。另外的实验显示这种相互作用是高度特异性的,因为pfHP1不结合仅在甲基化赖氨酸残基(H3K4(Me2))的位置上不同的另一个二甲基化肽,也不结合乙酰化的肽(H3K9(Ac))或未修饰的肽(H3)(图4B)。我们还可以证明,这种相互作用的特异性是保守的,因为也有来自 D的HP1。 melanogaster (dmHP1)显示相同的结合偏好(图4C和4D)。然而,与H3K9(Me2 / 3)相互作用的KD高约7倍(31.12±0.48μM)。 />
      4. 这些实验也证明用MST确定的结合等温线是高度可靠的。来自结合实验的几个单独重复的标准偏差是低的,并且确定的K D值是高度可重现的(图4A和4C)。此外,pfHP1和H3K9(Me2.4)(4.36μM)之间相互作用的K d值非常吻合由ITC确定的7μM(Jacobs和Khorasanizadeh,2002)。这表明MST非常适合于研究表观遗传系统中的相互作用。重要的是,测量可以在溶液中进行,样品消耗非常少(在使用真核蛋白质或合成肽时需要考虑的重要一点),也可以在生物液体中进行。因此,这项技术可以快速,简单,灵活地表征各种组蛋白 - 读数蛋白相互作用。


        图4.恶性疟原虫与恶性疟原虫之间相互作用的MST分析。黑色素瘤 HP1和使用无标记MST技术的不同组蛋白肽。来自恶性疟原虫(pfHP1)和 D的HP1蛋白。黑腹果蝇(dmHP1)作为本例中的目标。在这个例子中,没有必要附加荧光标签的目标,因为这两种蛋白具有足够高的摩尔消光系数来分析Monolith NT.LabelFree设备上的相互作用。一个先决条件是组蛋白肽不含色氨酸和酪氨酸残基(在300nm以上没有荧光)。

笔记

  1. 检测吸附和毛细血管粘连的效果和故障排除
    毛细管扫描可以检测目标和毛细管玻璃表面之间发生的粘附或吸附效应。不规则的峰形(如图2A,毛细管1-3),如U形或扁平的峰,表示粘附或吸附效应。在许多情况下,改变毛细管类型(标准处理,优质涂层或疏水)可以防止它们,导致形成规则的峰(图2A,毛细管4-6)。如果毛细管类型的改变不能增强毛细管扫描峰形,缓冲条件的变化或添加洗涤剂如吐温(0.005-0.1%)或Pluronic F-127(0.01-0.1%)可防止粘附和吸附。通常,在测量全部16个MST混合物之前,应该用一个MST混合物进行毛细管测试,以便找到不发生粘附和吸附的条件。
    这个阶段的MST实验对于高质量的数据质量至关重要
  2. 检测荧光效应和故障排除
    1. 最初的毛细管扫描还提供了有关可能的移液错误或配体依赖性荧光发射变化的信息。移液误差反映在毛细管峰的随机高度差异中。高移液精度是强制性的,以避免这种错误。另外,应该避免缓冲液稀释效应,即,用于制备稀释步骤的缓冲液应该与配体工作溶液的缓冲液完全相同。单个毛细血管之间荧光读数的巨大差异也可能是样本聚集的标志。聚集体的特征在于更高的荧光密度,从而在进入光学读出焦点时增加荧光读数。
    2. 然而,与配体浓度相关的荧光信号的系统变化暗示了配体可能的荧光增强或猝灭作用。为了排除其他可能性,如非特异性荧光降低,对于这些系统变化,进行了SD检验:如果荧光的系统变化实际上是配体依赖性的,则该变化在变性条件下不可检测到,并且荧光信号对于所有毛细血管应该是相同的。然而,如果荧光信号在变性条件下仍然显示出偏差,原因可能是荧光标记的分子由于吸附,粘附或聚集效应而损失。标准的SD-测试如下进行:将10μl第一个MST混合物和10μl最后一个MST混合物各自转移到含有10μl2×SD混合物(4%SDS,40mM DTT)的新鲜PCR管中, 。混合后,样品在95℃孵育5分钟以确保变性。新鲜的毛细血管被填满,荧光强度被记录在与之前相同的设置。
  3. 检测聚合效果和故障排除
    样品聚集和/或降水可以很容易地在MST迹线中检测为隆起和尖峰(图2B,左图)。没有聚合的样品通常显示平滑的MST迹线(图2B,右图)。如果在样品中发生聚集,则改变毛细管类型,缓冲条件和添加剂如洗涤剂(0.005-0.1%Tween-20,0.01-0.1%Pluronic F-127或类似物)或BSA(> 0.5mg / ml ),pH条件和盐浓度可以优化分子的溶解度。通过离心测量可以除去大的聚集体(至少10分钟,14,000×g克)。为了确保最佳的数据质量,MST迹线应该类似于图2B右侧面板中显示的迹线。
  4. 数据分析和曲线拟合
    1. 原则上,可以根据不同的潜在物理现象评估MST迹线。温度跃变(或T跳跃)描述了如果荧光发射随温度变化而发生突然变化,这对于不同荧光团而言通常是特征性的。 T跳跃受到配体在荧光团附近的结合的高度影响。 MST曲线的较慢热泳相受到分子沿感应温度梯度的移动的影响,因此对所研究分子的大小,电荷和水合壳的变化高度敏感。
    2. 在MO.Affinity分析软件的“专家”分析模式中,用户可以改变T跳跃或热泳评估的时间点。如果样品中发生聚集,并且上述说明不能解决这些问题,则有可能使用热泳阶段的早期部分,其中聚集效应可能小于后面的部分。检查热泳相的早期部分也可以提高数据质量,因为毛细血管内的对流效应不明显。
    3. MO.Affinity分析软件为拟合剂量反应曲线提供了两种选择。 “ D ”模型基于质量作用定律:



      其中,T 是标记的靶标,而 L 是非荧光配体。
      依赖于滴定配体浓度的荧光信号( f )可以是计算如下:



      其中, 和 是在未绑定状态和绑定状态下的荧光值,以及和 c T 是形成的复合物的浓度和荧光标记的目标的固定浓度。
      从分数边界值 D 可以派生出来:



      其中,K 是解离常数。
      请注意,此拟合模型仅适用于描述目标T与具有一个特定结合位点的配体L或具有相同亲和力的多个结合位点之间的1:1相互作用的数据。 “Hill”模型允许确定交互的EC50值:



      在这种情况下,分数界限如下:




      其中, 是滴定的非标记配体的浓度。
      请注意,“希尔”模型通常用于与协同绑定的交互。它不应该用于拟合可以用群众行动规律来清楚解释的数据。此外,“希尔”模型仅确定分子相互作用的EC 50值,其中50%的目标分子结合的配体的浓度。知道EC50值不直接对应于解离常数K 是很重要的。
      对于一个特定的实验来说,这更多的是一种明显的亲和力,它取决于所使用的浓度和条件
  5. 检测低荧光团浓度
    低于1nM的标记靶的浓度通常需要高的激发光强度(Monolith NT.115微微上的LED功率> 75%(或“高”))。如此高的强度会导致荧光团的明显的漂白,并因此给数据带来额外的噪音。 NanoTemper Technologies提供的抗漂白剂套件可以帮助减少这些影响。
  6. 关于温度梯度
    温度梯度跨度为2°C,MST功率为20%,温度梯度为6°C,MST功率为80%。加热样品的总体积是2 nl。建议通过使用单个毛细管和不同的MST功率设置(例如20%,40%和80%)进行多次测量,在全结合实验之前确定最佳的MST功率。

食谱

  1. NaP缓冲区
    10mM磷酸钠pH 7.0
    1 mM EDTA
    1 mM DTT
    0.5毫米PMSF
  2. MST-T缓冲区
    50mM Tris-HCl pH 7.8
    150 mM NaCl
    10mM MgCl 2•/ 2 0.05%Tween-20

致谢

我们感谢Corinna Kuttenberger和Clemens Entzian提供专家科学和技术援助。

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引用:Plach, M. G., Grasser, K. D. and Schubert, T. (2017). MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes. Bio-protocol 7(23): e2632. DOI: 10.21769/BioProtoc.2632.
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