Name: measuring biomolecular dsc profiles with thermolabile ligands to rapidly characterize folding and binding interactions
Uploaded: 2017-11-21T12:30:00.000+00:00
Duration: 9 min 15 s
Description: Watch this Scientific Journal Video about Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions at JoVE.com

R. W. H. V was supported by the McGill Natural Sciences and Engineering Research Council of Canada (NSERC) Training Program in Bionanomachines. A. K. M. and P. E. J. were supported by NSERC grants 327028-09 (A. K. M) and 238562 (P. E. J.).

1. Sample Preparation
<ol>
	<li>Purify the desired biomolecule13. 
	NOTE: This protocol uses purchased cocaine-binding DNA aptamer MN4 after exchanging against 2 M NaCl three times followed by three rounds of deionized water using a centrifugal filter with a 3 kDa molecular weight cut-off membrane.</li>
	<li>Synthesize and purify, or purchase the desired thermolabile ligand13. 
	NOTE: MN4 binds the thermolabile ligand cocaine. MN4 also binds quinine, which is used as a negative control for ligand thermolability at these experimental temperatures.</li>
	<li>Prepare buffers for dialysis of the purified biomolecule and dissolution of ligands (20 mM sodium phosphate and 140 mM NaCl buffer, pH 7.4, for MN4 and the ligands used here).</li>
	<li>Dialyze the biomolecule against at least 2 L of buffer using dialysis tubing with 0.5 - 1.0 kDa cut-off.</li>
	<li>Filter the final buffer (referred to as working buffer) through a 0.2 &#181;m filter that has been thoroughly equilibrated with buffer.</li>
	<li>Weigh out the desired masses of the ligands and dissolve them in filtered working buffer. If the desired ligand concentrations require masses that are too small to accurately weigh, make a concentrated ligand stock solution (10x for example). 
	NOTE: It is crucial that all DSC experiments utilize the same working buffer for the sample and ligand, i.e., never perform an experiment where the ligand is dissolved in a different batch of working buffer than the biomolecule as this will cause buffer mismatch artifacts in the data.</li>
	<li>Filter the biomolecule stock solution through a 0.2 &#181;m filter that has been thoroughly equilibrated with working buffer.</li>
	<li>Determine the biomolecule concentration by absorbance measurements (260 nm for nucleic acids like MN4 and 280 nm for proteins).</li>
	<li>Store the prepared biomolecule and ligand in a 4 &#176;C refrigerator (suitable for MN4 and the ligands used here), or at -20 or -80 &#176;C if the biomolecule and ligands tolerate freezing and long-term storage is required. Degas the buffer, biomolecule, and ligand solutions in a table top degasser (see Table of Materials) prior to loading into the DSC. 
	NOTE: Degassing helps to prevent bubble formation in the DSC at higher temperatures. Bubbles cause signal artifacts that obscure DSC peak shapes and baselines.</li>
</ol>
2. DSC Preparation
<ol>
	<li>Unscrew the pressure handle from the DSC (see Table of Materials).</li>
	<li>Run silicon tubing from the working buffer and attach it to the front flange (metal opening) of the reference capillary.</li>
	<li>Create a bridge between the reference and sample capillaries by connecting the rear reference flange to the front sample flange.</li>
	<li>Attach a piece of silicon tubing to the rear sample flange that runs to a waste flask with a vacuum line attached.</li>
	<li>Turn on the vacuum line to flush the DSC with 200 mL of working buffer.</li>
	<li>Load the reference capillary of the DSC with working buffer. Attach roughly 3 - 5 cm sections of silicon tubing to the reference capillary flanges.</li>
	<li>Insert a 1 mL pipette tip into the rear flange&#39;s silicon tubing. Draw 0.8 mL of working buffer with a pipette and insert the pipette tip with buffer into the front reference flange&#39;s silicon tubing.</li>
	<li>Gently press the pipette plunger down to pass the working buffer through the front silicon tubing into the reference capillary and up into the rear flange&#39;s attached pipette tip. Press down the pipette plunger until the working buffer level reaches just above the front silicon tubing, then release the pipette plunger until the working buffer level reaches just above the rear silicon tubing.
	<ol>
		<li>Repeat passing the working buffer back and forth to purge the volume in the reference capillary of bubbles. 
		NOTE: Usually, 10 passes of the solution back and forth are sufficient to clear any bubbles.</li>
	</ol>
	</li>
	<li>Cap the rear pipette tip with the thumb and gently pull up on the rear pipette tip and front pipette to remove them from the reference flanges with the silicon tubing attached.</li>
	<li>Load the sample capillary with working buffer as in steps 2.6 - 2.9. Place a black plastic cap on the rear reference and sample flanges, leaving the front flanges uncovered.</li>
	<li>Attach the pressure handle.</li>
	<li>Open the DSC software (see Table of Materials) and pressurize the instrument by clicking the red up arrow at the top of the interface once the power reading has stabilized; the DSC power is indicated in a box at the top right of the interface along with the instrument temperature and pressure reading. 
	NOTE: Monitor the power reading as the DSC pressurizes. Changes in power of more than ~ 10 &#181;W indicate bubble formation in the capillaries, which can cause artifacts in the data. The solutions must be removed and degassed further before continuing.</li>
	<li>Equilibrate the DSC with working buffer by performing a forward and reverse scan. In the &#34;Experimental Method&#34; tab on the left side of the screen, ensure that the &#34;Scanning&#34; option is selected to run the DSC in temperature scanning mode. 
	NOTE: Experimental parameters are the temperature scanning range, scan rate, low and high temperature equilibration time, and number of scans.
	<ol>
		<li>In the &#34;Temperature Parameters&#34; inset under the &#34;Experimental Method&#34; tab, click the button for &#34;Heating&#34;. Enter 1 and 100 &#176;C for the lower and upper experimental temperatures, 1 &#176;C/min for the scan rate, and 60 s for the equilibration period.</li>
		<li>Click the &#34;Add Series&#34; button under the input field for the equilibration period. Enter 2 into the &#34;Steps to add&#34; field in the popup window (for one heating and one cooling scan) and check the &#34;Alternate Heating/Cooling&#34; box. Click &#34;OK&#34;; the added scans appear in the lower portion of the interface. Check that the parameters for each scan are as desired.</li>
		<li>Start the experiment by clicking the green &#34;play&#34; button at the top of the interface. Navigate to the desired folder and input a file name for saving the experiment in the popup window. View the experiment progress by clicking the &#34;Data&#34; tab to the right of the &#34;Experiment Method&#34; tab.</li>
	</ol>
	</li>
</ol>
3. Collecting Thermolabile Ligand DSC Datasets
NOTE: The minimal procedure consists of five experiments: buffer reference experiments with and without ligand (used for baseline subtraction, see Discussion), sample experiments with the free biomolecule, the ligand-bound biomolecule, and the ligand-bound biomolecule with a longer high temperature equilibration period.
<ol>
	<li>Run reference experiments for baseline subtraction of the sample data. Reload the DSC with working buffer in both capillaries and collect multiple forward and reverse scans over a suitable temperature range at 1 &#176;C min-1 with an upper (high temperature) equilibration time of 120 s.
	<ol>
		<li>Delete the previous buffer equilibration scans from the lower portion of the interface by highlighting each individually and clicking the red X to the middle right of the interface. Add the new scans by clicking the &#34;Add Series&#34; button, entering 20 in the field for &#34;Steps to add&#34;, and checking the &#34;Alternate Heating/Cooling&#34; box. Click OK and run the experiment by clicking the green play button as above.</li>
		<li>Repeat steps 3.1 - 3.1.1 with working buffer containing the desired concentration of ligand in both capillaries to obtain the reference experiments for the ligand (collect two separate experiments using 120 s and 600 s high temperature equilibration times respectively, to be used in acquiring the rate constant for thermolabile ligand conversion). 
		NOTE: The scan rate used here ensures that the biomolecule in subsequent experiments is at thermal equilibrium in the forward and reverse scans (see Discussion). Scan rates &#60; 0.1 - 0.2 &#176;C min-1 lead to noisy thermograms and are not applicable in DSC experiments. The temperatures should extend from well below the melting temperature of the free biomolecule to well above the melting temperature of ligand-saturated biomolecule (~ 20 - 80 &#176;C for MN4). Verify reproducibility of the scans (for example, 10 forward and 10 reverse scans for 20 total is sufficient).</li>
		<li>If using multiple ligands (such as cocaine and quinine), flush the DSC with 200 mL of working buffer (repeat from&#160;step 2.5) between runs in order to remove ligand from the capillaries and prevent cross-contamination. 
		NOTE: It is helpful to perform a replicate experiment on the free biomolecule after a ligand-bound run in order to check if the previous ligand adsorbs strongly to the capillary walls and is not adequately removed with buffer flushing. If the thermograms for the free biomolecule appear to be shifted to a larger magnitude and higher denaturation temperature after the ligand-bound experiment, it is likely that the previous ligand is still present in the calorimeter after flushing. Remove the adsorbed ligand by incubating the capillaries with 20% Contrad-70 for 1 h at 60 &#176;C with the plastic caps and pressure handle off. In the DSC software, change the experimental mode to &#34;Isothermal&#34; under the Experimental Method tab. Choose 3,600 s for the duration and 60 &#176;C for the isothermal temperature, with zero entered for the equilibration time. Click &#34;Add to Experimental Method&#34;. The isothermal experiment appears in the lower part of the screen. After completion, flush the instrument with 2 L deionized water and repeat from step 2.5.</li>
	</ol>
	</li>
	<li>Run sample experiments using the same DSC loading procedure and experimental parameters as the reference scans. For the free biomolecule data set, ensure that the reference capillary contains the working buffer while the sample capillary contains the free biomolecule at the desired concentration in working buffer.
	<ol>
		<li>For the ligand-bound experiments, ensure that the ligand is in the working buffer in the reference capillary, and the biomolecule plus ligand are in working buffer in the sample capillary. Flush the system between additions of different ligands as in step 2.5.</li>
	</ol>
	</li>
	<li>Perform one additional experiment with the biomolecule bound to the thermolabile ligand where the high temperature equilibration period is increased to 600 s and all other experimental parameters are the same as step 3.2.1. 
	NOTE: The duration of the high temperature equilibration period for the second ligand-bound experiment is simply chosen to ensure that the ligand is more rapidly depleted than the short equilibration time experiment. If the ligand-bound peaks from the first experiment decay slowly as a function of scan number (e.g., the differences in successive peak maxima are &#8804; 0.5 &#176;C), estimate 10- to 20-fold increases in the high temperature equilibration period in order to adequately deplete the ligand during the second experiment. The accurate calculation of the rate constant for ligand conversion requires that the second experiment has swifter depletion of the ligand relative to the first. The ligand concentrations extracted from the global analysis of the two experiments will be similar and therefore unusable if the ligand depletion is not sufficiently accelerated in the second experiment.</li>
</ol>
4. Data Processing
<ol>
	<li>Open the DSC experiment files in the DSC data analysis software (see Table of Materials) and export the raw power data as spreadsheets.</li>
	<li>Import the spreadsheets containing the raw power data into software for data fitting.</li>
	<li>Baseline subtract the sample data by subtracting the buffer power data from the free and ligand-bound biomolecule experiments. 
	NOTE: For the thermolabile ligand experiment, the concentration of initial ligand is decreasing with each scan. Therefore, it is ideal to subtract the buffer scan 1 from sample scan 1, and so on. We have found that the buffer scans with cocaine do not change appreciably as the ligand conversion proceeds and therefore a single thermolabile ligand buffer scan can be used to subtract all of the thermolabile ligand-bound data.</li>
	<li>Convert the baseline-subtracted sample power data to heat capacity. 
	NOTE: The conversion requires the biomolecule&#39;s partial specific volume, which can be estimated14,15,16. The equation for converting power to heat capacity has been described previously17.</li>
</ol>
5. Data Analysis
<ol>
	<li>Globally fit the short equilibration time thermolabile ligand-bound heat capacity dataset with a single set of baseline, ligand concentration, folding, and ligand binding parameters as described previously4.</li>
	<li>Repeat the global fit for the long equilibration time dataset in order to calculate the rate constant for thermolabile ligand conversion as described previously4.</li>
</ol>

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The authors declare no conflicts of interest.

Modifications and troubleshooting
The details of the global fitting analysis used in Figure 1 and Figure 2 have been described previously4. Here, we outline practical aspects of performing and analyzing DSC binding experiments with thermolabile ligands. Note that a DSC baseline obtained for the thermolabile ligand alone is subtracted from the ligand + biomolecule dataset, effectively cancelling ou...

Differential scanning calorimetry (DSC) is a powerful method for quantitating biomolecular binding and folding interactions1,2,3. The strengths of DSC include its ability to elucidate binding and folding mechanisms, and to yield the corresponding thermodynamic parameters2,3. Furthermore, DSC can be performed in solution under near-physiological conditions and does not require labeling of the biomolecule or ligand, e.g., with fluorophores, spin-labels or nuclear isotopes4. The instrument scans in temperature, measuring the amount of heat required to denature the biomolecule in the presence and absence of ligand. The resulting thermograms are used to extract the thermodynamic parameters governing the ligand binding and folding processes. The information provided by DSC or other thermodynamic techniques is critical to guiding the design of drugs targeting biomolecules1,5,6,7,8. However, the repeated scanning to high temperatures (~ 60 - 100 &#176;C) can be problematic. For example, many pharmaceutically important compounds undergo rearrangement or decomposition upon sustained exposure to high temperatures9,10,11, i.e., they are thermolabile. Examination of binding interactions by DSC typically requires multiple forward and reverse scans in order to verify the reproducibility of the thermogram for thermodynamic analyses12. Thermal conversion of an initial ligand to a secondary form with altered binding characteristics leads to pronounced differences in the shape and position of successive thermograms, since the concentration of the initial ligand decreases with each scan while the thermal conversion products accumulate. These datasets are not amenable to traditional analyses.
We have recently developed a global fitting method for thermolabile ligand DSC datasets that yields the complete set of thermodynamic parameters governing the biomolecular folding and binding interactions from a single ligand-bound experiment referenced to the requisite thermogram for the free biomolecule4. The analysis reduces the experimental time and sample required by ~ 10-fold compared to standard DSC approaches. We have accounted for ligand thermal conversion by assuming this happens during the high temperature portion of each scan where the thermogram does not depend on ligand concentration. Therefore, the ligand concentration is a constant within the portion of the thermogram that is used to extract thermodynamic parameters. We additionally demonstrated how the rate constant for ligand thermal conversion can be obtained by performing one supplementary experiment with a longer high temperature equilibration period. For systems where ligand thermal conversion is less temperature-dependent (i.e., occurring appreciably at all temperatures), the analysis can be modified to include variable ligand concentrations. Here we demonstrate this procedure for the DNA aptamer MN4 in the presence of the thermolabile ligand cocaine, which rapidly converts to benzoylecgonine at high temperatures (&#62;60 &#176;C). Quinine is used as a negative control for ligand thermolability since it does not undergo conversion at these experimental temperatures and also binds to MN4. We describe the acquisition of thermolabile ligand DSC datasets and their analysis yielding thermodynamic and kinetic parameters of the folding, binding, and ligand conversion processes.

<table><tbody><tr><td>Sodium chloride</td><td>Chem Impex</td><td>#00829</td><td></td></tr><tr><td>Sodium phosphate monobasic dihydrate</td><td>Sigma Aldrich</td><td>71502</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S9763</td><td></td></tr><tr><td>Deioinized water for molecular biology</td><td>Millipore</td><td>H20MB1001</td><td></td></tr><tr><td>0.2 micron sterile syringe filters</td><td>VWR</td><td>CA28145-477</td><td></td></tr><tr><td>3 kDa centrifugal filters</td><td>Millipore</td><td>UFC900324</td><td></td></tr><tr><td>Dialysis tubing 0.5-1.0 kDa cutoff</td><td>Spectrum Laboratories</td><td>131048</td><td></td></tr><tr><td>Silicon tubing</td><td>VWR</td><td>89068-474</td><td></td></tr><tr><td>Plastic DSC flange caps</td><td>TA Instruments</td><td>6111</td><td></td></tr><tr><td>DNA aptamer MN4</td><td>Integrated DNA Technologies</td><td>https://www.idtdna.com/site/order/menu</td><td></td></tr><tr><td>Cocaine</td><td>Sigma Aldrich</td><td>C008</td><td></td></tr><tr><td>Quinine</td><td>Sigma Aldrich</td><td>22620</td><td></td></tr><tr><td>NanoDSC-III microcalorimeter</td><td>TA Instruments</td><td>http://www.tainstruments.com/nanodsc/</td><td></td></tr><tr><td>DSCRun software</td><td>TA Instruments</td><td>http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/</td><td></td></tr><tr><td>NanoAnalyze software</td><td>TA Instruments</td><td>http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/</td><td></td></tr><tr><td>Contrad-70</td><td>VWR</td><td>89233-152</td><td></td></tr></tbody></table>

measuring biomolecular dsc profiles with thermolabile ligands to rapidly characterize folding and binding interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

Representative data for the thermolabile ligand DSC are shown in Figure 1. The position and height of the thermolabile ligand-bound peak successively shifts down towards that of the unbound biomolecule as the thermolabile ligand is depleted with each scan (Figure 1a). The free denaturation profile is used as a reference for the endpoint of thermolabile ligand conversion (Figure 1b). Data for MN4 boun...

Watch this Scientific Journal Video about Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions at JoVE.com

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

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8.2K Views.  McGill University. We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

Video: Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Bioengineering

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

High-Throughput Thermal Shift Assays for Protein-Ligand Interactions

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. 
Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Biology

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

High-Sensitivity Detection of Compound-Protein Binding Using Fluorescence

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...