A.S. is a W.W. Caruth, Jr. Scholar in Biomedical Research, Cancer Prevention and Research Institute of Texas (CPRIT) Scholar, and Charles and Jane Pak Center for Mineral Metabolism and Clinical Research Faculty Scholar. This work was supported by NIH Grant K01DK123194 (A.S.), CPRIT Grant RR190106 (A.S.), Welch (I-2046-20200401) and Welch (I-2046-20230405).

1. Experimental setup
<ol>
	<li>Use a thermal cycler that can detect fluorescence, such as an RT-PCR instrument, to perform the described protocol. If using SYPRO Orange, as described in this protocol, use a scanning mode that allows for excitation around 470 nm and emission detection around 570 nm. Program the thermocycler to hold the sample at 20 &#176;C for 2 min, followed by an increase in temperature of 0.5 &#176;C/1 min up to 95 &#176;C; measure fluorescence intensity every 1 &#176;C. 
	NOTE: We recommend an optional step at the end of the cycle to return the sample block to 20 &#176;C (Figure 1).</li>
	<li>Each reaction contains four components: Buffer, protein of interest, additive, and extrinsic fluorescent dye. Design the experiment to include controls such as no protein, no dye, protein without additives, and additives alone to determine any background fluorescence that may influence the interpretation of the results. If available, include a positive control such as an additive known to bind and stabilize the protein of interest.</li>
	<li>Use purified protein for the assay. To follow this example, use E. coli SelO, expressed and purified as previously described25,29. Briefly, express His-sumo tagged SelO (E. coli SelO ppSumo) in Rosetta DE3 cells and purify it using Ni2+-NTA affinity. Cleave the His-sumo tag using Ulp protease and purify SelO further by gel filtration chromatography.</li>
	<li>The fluorescence of SYPRO Orange will depend on its interaction with the protein of interest. As these interactions will vary from protein to protein, optimize the protein and dye concentration for obtaining the maximum signal to noise of fluorescence signal. 
	NOTE: SYPRO Orange is not compatible with some proteins and additives such as detergents11,30. Additional extrinsic dyes listed in 12 may be screened as necessary.</li>
</ol>
2. Determination of the optimal dye concentration (Figure 2A)
<ol>
	<li>Prepare 20 &#181;L&#160;dilutions of 50x, 40x, 30x, 20x, and 10x SYPRO Orange from the stock solution, which is provided at 5,000x in DMSO.</li>
	<li>Dispense 8 &#181;L of 6.25 &#181;M protein diluted in TSA buffer into six separate wells of a 384-well PCR plate. 
	NOTE: In our example, we used the following TSA buffer to perform the dilutions: 10 mM Tris pH 8, 150 mM NaCl, and 1 mM DTT. Samples may be run in triplicate to improve the reliability of outcomes.</li>
	<li>Add 1 &#181;L of TSA buffer to each well. 
	NOTE: This volume of buffer will be replaced with the addition of small molecules after optimization of dye and protein concentration.</li>
	<li>Add 1 &#181;L of each serial dilution of SYPRO Orange dye solution to each well. Add 1 &#181;L of buffer without dye to the final well for the no dye control.</li>
	<li>Cover the plate with optically clear adhesive film.</li>
	<li>Briefly spin down the plate at 1,000 &#215; g for 1 min in a centrifuge equipped with a PCR plate adapter.</li>
	<li>Place the plate in an RT-PCR machine and start the thermocycling protocol described in step 1.1.</li>
</ol>
3. Determination of the optimal protein concentration (Figure 2B)
<ol>
	<li>Prepare 20 &#181;L of serial dilutions of 25 &#181;M, 12.5 &#181;M, 6.25 &#181;M, 3.125 &#181;M, and 1.56 &#181;M of protein using TSA buffer (10 mM Tris pH 8, 150 mM NaCl, and 1 mM DTT).</li>
	<li>Dispense 8 &#181;L of serial dilutions of the protein into five separate wells of a 384-well plate.</li>
	<li>Dispense buffer only in the 6th well as a no protein control.</li>
	<li>Add 1 &#181;L of TSA buffer to each well. 
	NOTE: This volume of buffer will be replaced with the addition of small molecules after optimization of dye and protein concentration.</li>
	<li>Add 1 &#181;L of each 50x SYPRO Orange dye solution to each well.</li>
	<li>Cover the plate with optically clear adhesive film.</li>
	<li>Briefly spin down the plate at 1,000 &#215; g for 1 min in a centrifuge equipped with a PCR plate adapter.</li>
	<li>Place the plate in an RT-PCR machine and start the thermocycling protocol described in step 1.1.</li>
</ol>
4. Setting up a TSA experiment (Figure 3A)
NOTE: After optimization of the protein and SYPRO Orange dye concentrations, the assay can be performed with the addition of the desired small molecules to analyze binding.
<ol>
	<li>Define the number of conditions by considering the replicates and adequate controls. 
	NOTE: For example, we will test the binding of SelO to eight conditions in triplicate. Including the controls (no additive, no protein, and no dye), we have 11 reactions x 3 (triplicate) = 33 total reactions.</li>
	<li>Based on the optimal concentration of protein and dye observed for SelO, make sure that each reaction contains 8 &#181;L of 6.25 &#181;M protein, 1 &#181;L of additive, and 1 &#181;L of 50x SYPRO Orange dye for a total of 10 &#181;L reaction volume. Thus, the final concentration used is 5 &#181;M protein and 5x SYPRO Orange dye.</li>
	<li>Prepare 288 &#181;L of 6.25 &#181;M protein solution using TSA buffer. This volume of protein working solution accounts for 36 reactions (33 reactions with an additional 10% for variations in pipetting).</li>
	<li>Dispense 8 &#181;L of 6.25 &#181;M protein into separate wells of a 384-well plate. Dispense buffer only for the no protein control.</li>
	<li>Add 1 &#181;L of small molecules at 10x concentration to achieve final 1x. To follow this example, add 1 &#181;L of 20 mM MgCl2 to achieve a final concentration of 2 mM. Dispense buffer only for the no additive control.</li>
	<li>Add 1 &#181;L of each 50x SYPRO Orange dye solution to each well.</li>
	<li>Cover the plate with optically clear adhesive film.</li>
	<li>Briefly spin down the plate at 1,000 &#215; g for 1 min in a centrifuge equipped with a PCR plate adapter.</li>
	<li>Place the plate in an RT-PCR machine and start the thermocycling protocol described in step 1.1.</li>
</ol>
5. Data analysis
<ol>
	<li>Export data from the RT-PCR machine for relative fluorescence units (RFU) with respect to temperature (&#176;C).</li>
	<li>Use the software of choice to extract and plot data points up to the highest intensity measured for each melting curve. Importantly, confirm that the no protein and no dye controls exhibit low background fluorescence without temperature-dependent increases in fluorescence (Figure 3A,B and Supplemental Table S1). Perform data analysis using freely accessible software such as MoltenProt31 or DSFWorld32.</li>
	<li>Determine the melting temperature by using Boltzmann Sigmoidal Fit for the data. The melting temperature (Tm) is defined as the temperature at which 50% denaturation or half maximal intensity is observed (Figure 3C).</li>
	<li>A thermal shift in the melting curve may suggest stabilizing or destabilizing additives. To calculate the change of melting temperature (Tm), use the formula: Tm= Tm additive- Tm buffer. A positive value describes a stabilizing condition or interacting additive; a negative value describes a destabilizing condition (Figure 3D).</li>
</ol>

High-Throughput Thermal Shift Assays for Protein-Ligand Interactions

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	<li>Dunham, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+genes:+Time+to+follow+the+roads+less+traveled.">Human genes: Time to follow the roads less traveled.</a> PLoS Biol. 16 (9), e3000034 (2018).</li><li>Kustatscher, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+invitation+to+the+understudied+proteins+initiative.">An open invitation to the understudied proteins initiative.</a> Nat Biotechnol. 40 (6), 815-817 (2022).</li><li>Jones, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+genomics+consortium:+A+knowledge+platform+for+drug+discovery:+A+summary.">The structural genomics consortium: A knowledge platform for drug discovery: A summary.</a> Rand Health Q. 4 (3), 19 (2014).</li><li>Oberg, N., Zallot, R., Gerlt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efi-est,+efi-gnt,+and+efi-cgfp:+Enzyme+function+initiative+(efi)+web+resource+for+genomic+enzymology+tools.">Efi-est, efi-gnt, and efi-cgfp: Enzyme function initiative (efi) web resource for genomic enzymology tools.</a> J Mol Biol. 435 (14), 168018 (2023).</li><li>Pace, C. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+stability+and+ligand+interactions+by+thermal+shift+assay.">Analysis of protein stability and ligand interactions by thermal shift assay.</a> Curr Protoc Protein Sci. 79, 21-29 (2015).</li><li>Niesen, F. H., Berglund, H., Vedadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+differential+scanning+fluorimetry+to+detect+ligand+interactions+that+promote+protein+stability.">The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.</a> Nat Protoc. 2 (9), 2212-2221 (2007).</li><li>Wu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformationally+responsive+dyes+enable+protein-adaptive+differential+scanning+fluorimetry.">Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry.</a> bioRxiv. , (2023).</li><li>Wu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+performing+and+optimizing+differential+scanning+fluorimetry+experiments.">Protocol for performing and optimizing differential scanning fluorimetry experiments.</a> STAR Protoc. 4 (4), 102688 (2023).</li><li>Gao, K., Oerlemans, R., Groves, M. 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The authors declare no competing interests.

The Thermal Shift Assay (TSA) serves as an efficient method for screening protein-ligand interactions, including those with cofactors and inhibitors. In this protocol, we used TSA to measure the binding of the pseudokinase SelO to nucleotides and divalent cations. Our findings show that SelO exhibits increased thermal stability in the presence of ATP and Mg2+/Mn2+. This observation aligns with previous reports indicating that SelO homologs from E. coli, S. cerevisiae, and H. sapiens</em...

Much of the human proteome remains poorly characterized. The &#34;streetlight effect&#34; described by Dunham and Kustatscher et al. refers to the phenomenon where extensively researched proteins receive more attention, leaving understudied proteins overlooked1,2. Factors contributing to this effect include the biological importance and disease relevance of certain proteins. Moreover, prior research that offers foundational knowledge, as well as the availability of tools for functional analysis, further stimulates research on highly studied proteins1,2. Projects such as the Understudied Proteins Initiative, Structural Genomics Consortium, and Enzyme Function Initiative aim to characterize understudied proteins using structural and functional proteomics2,3,4. A complementary approach to identifying the molecular functions of understudied proteins is to examine the interactions of these proteins with small molecules to gain valuable insights into the regulation and substrate specificity.
The binding of small molecules can increase the thermal stability of a protein5. This phenomenon, known as ligand-induced conformation stabilization, often results in an increase in the melting temperature of a protein, providing a measurable indication of ligand binding6. The thermal stability of a protein can be measured using Differential Scanning Fluorimetry (DSF), also known as Thermofluor, or Thermal Shift Assay (TSA)7,8,9,10. In TSA, a protein sample is subjected to increasing temperatures in the presence of an environmentally sensitive dye6. As the protein unfolds and denatures, the dye binds to the exposed hydrophobic regions of the protein and emits fluorescence. Extrinsic fluorescent dyes, or environmentally sensitive dyes, lack intrinsic fluorescence and instead fluoresce upon interacting with their target molecule9,11,12. The most commonly used dye, SYPRO Orange, offers several advantages such as enhanced stability and minimal background fluorescence8,9,12. Notably, SYPRO Orange is compatible with Real-Time Polymerase Chain Reaction (RT-PCR) systems given its excitation and emission wavelengths around 470 nm and 570 nm, respectively. This unique feature allows for high-throughput, accurate, and sensitive measurements that are compatible with fluorescence detection systems commonly used in RT-PCR systems8.
TSA is a versatile tool for investigating protein interactions with potential cofactors or drugs and for identifying stabilizing conditions for crystallography. Some of its advantages over other screening methods are its relative simplicity of setup and high throughput with 96- or 384-well plates13,14,15. The development of this technique has been transformative for drug discovery studies, offering convenience and enabling the study of diverse additives such as ions, ligands, and drugs6,16. Moreover, the adaptability of TSA has encouraged scientists to expand its utility, facilitating the measurement of binding affinities alongside the screening assays17,18. TSA is an effective tool in structural biology studies to identify conditions that promote the stabilization of the protein of interest for crystallization19. Thus, its flexibility and relative ease have positioned TSA as a cornerstone technique in characterizing proteins. Here, we will use TSA to analyze the binding of metals and nucleotide to the understudied pseudokinase, Selenoprotein O (SelO), as an example.
Kinases are one of the most targeted protein families in drug discovery20. Approximately 10% of human kinases are predicted to be inactive and named pseudokinases because they lack key catalytic residues required for catalysis21,22. Selenoprotein O (SelO) is an evolutionarily conserved pseudokinase that lacks the conserved aspartate in the catalytic HRD motif23. In eukaryotes, SelO localizes to the mitochondria and protects cells from oxidative stress24,25. Structural and biochemical analyses show that SelO transfers adenosine monophosphate (AMP) from ATP to protein substrates in a posttranslational modification known as AMPylation25. Recent studies indicate that enzymes that catalyze AMPylation may bind alternative nucleotides such as UTP in vitro26,27. Notably, studies have demonstrated that the SelO homolog from Salmonella typhimurium catalyzes protein UMPylation, or the transfer of UMP, in a manganese-dependent manner28. Given these intriguing observations, we test the binding of SelO to ATP and UTP in the presence of magnesium and manganese, serving as a representative example of TSA&#39;s applications. The following protocol can be readily adapted to optimize and examine the interactions of other proteins and additives of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td>Sigma Aldrich</td>
			<td>A2383-10G</td>
			<td>used for representative results but not required to perfom TSA</td>
		</tr>
		<tr>
			<td>Avanti J-15R with microplate carrier assembly</td>
			<td>Beckman Coulter</td>
			<td>C19416</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Opus 384 Real-time PCR system</td>
			<td>Bio-Rad</td>
			<td>12011452</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard-Shell 384-Well PCR Plates, thin wall, skirted, clear/white</td>
			<td>Bio-Rad</td>
			<td>HSP3805</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>M8266-100G</td>
			<td>used for representative results but not required to perfom TSA</td>
		</tr>
		<tr>
			<td>Manganese (II) chloride tetrahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M3634-500G</td>
			<td>used for representative results but not required to perfom TSA</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>SYPRO Orange Protein Gel stain</td>
			<td>Sigma Aldrich</td>
			<td>S5692-500UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine 5&#8242;-triphosphate trisodium salt hydrate</td>
			<td>Sigma Aldrich</td>
			<td>U6625-100MG</td>
			<td>used for representative results but not required to perfom TSA</td>
		</tr>
	</tbody>
</table>

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

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

We aim to study the biochemical activity of proteins with unknown functions. Co-factor binding is not only essential for the activity of some proteins, but may also alter their thermal stability. One practical application of this phenomenon lies in utilizing the change in thermal stability as measured by the protein's melting temperature to analyze ligand binding.Thermal shift assay is a versatile tool for investigating protein interactions with potential co-factors or drugs, and for identifying stabilizing conditions for crystallography. Some of its advantages over other screening methods are its relatively simple setup and high throughput with 96 or 384 well plates. Selenoprotein O, or SelO, is an evolutionarily conserved pseudokinase that transfers adenosine monophosphate from ATP to protein substrates in a post-translational modification known as AMPylation.In this protocol, we use TSA to analyze nucleotide and metal binding to SelO.

Eukaryotic SelO consists of an N-terminal mitochondrial targeting sequence, a kinase-like domain, and a highly conserved selenocysteine at the C-terminus of the protein23. This mitochondrial-resident enzyme encodes a pseudokinase domain that is conserved from bacteria to humans23. Structural analysis of the SelO homolog from Pseudomonas syringae revealed amino acid alterations in the active site that facilitate the binding of ATP in an inverted orientation in compa...

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 ...

utilizing-thermal-shift-assay-to-probe-substrate-binding-to

Research

JoVE Journal

Biochemistry

Thermal Shift Assay to Uncover Molecular Interactions of Selenoprotein O

To begin, prepare Selenoprotein solution Using thermal shift assay or TSA buffer. dispense the protein, small molecules, and SYPRO Orange dye to the appropriate wells of a 384-well plate. Cover the plate with optically clear adhesive film and briefly spin down the plate at 1, 000 g for one minute in a centrifuge.Then place the plate in a real-time PCR machine and program it to hold the sample at 20 degrees Celsius for two minutes, followed by a 0.5 degree increase in temperature per minute up to 95 degrees Celsius. Export the data from the RT PCR machine for relative fluorescence units or RFU with respect to temperature. Using the software of choice, extract and plot data points up to the highest intensity measured for each melting curve.Determine the melting temperature with the Boltzmann sigmoidal fit for the data to determine the melting temperature or Tm.The thermal denaturation curves indicated a four degrees Celsius increase Escherichia in the thermal stability of Escherichia coli SelO in the presence of ATP with magnesium ions and a 12 degrees Celsius rise in the same for ATP with manganese ions. However, there was no shift in thermal stability upon incubation with UTP, indicating that Escherichia coli SelO may not exhibit detectable binding to UTP. The no protein as well as the no dye controls had low fluorescence signal, which was not responsive to the temperature increase.

333 Views.  University of Texas Southwestern Medical Center.  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. 

High-Throughput Thermal Shift Assays for Protein-Ligand Interactions

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