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...

High-Throughput Thermal Shift Assays for Protein-Ligand Interactions

<ol>
	<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. N., Mcgrath, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+stabilization+of+lysozyme+to+thermal+and+guanidine+hydrochloride+denaturation.">Substrate stabilization of lysozyme to thermal and guanidine hydrochloride denaturation.</a> J Biol Chem. 255 (9), 3862-3865 (1980).</li><li>Pantoliano, M. W., 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=High-density+miniaturized+thermal+shift+assays+as+a+general+strategy+for+drug+discovery.">High-density miniaturized thermal shift assays as a general strategy for drug discovery.</a> J Biomol Screen. 6 (6), 429-440 (2001).</li><li>Huynh, K., Partch, C. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+applications+of+differential+scanning+fluorimetry+in+early-stage+drug+discovery.">Theory and applications of differential scanning fluorimetry in early-stage drug discovery.</a> Biophys Rev. 12 (1), 85-104 (2020).</li><li>Hawe, A., Sutter, M., Jiskoot, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+fluorescent+dyes+as+tools+for+protein+characterization.">Extrinsic fluorescent dyes as tools for protein characterization.</a> Pharm Res. 25 (7), 1487-1499 (2008).</li><li>Lucet, I. S., Murphy, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+for+broke:+Targeting+the+human+cancer+pseudokinome.">Going for broke: Targeting the human cancer pseudokinome.</a> Biochem J. 465 (2), 195-211 (2015).</li><li>Kung, J. E., Jura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+for+pharmacological+targeting+of+pseudokinases.">Prospects for pharmacological targeting of pseudokinases.</a> Nat Rev Drug Discov. 18 (7), 501-526 (2019).</li><li>Dudkiewicz, M., Szczepinska, T., Grynberg, M., Pawlowski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protein+kinase-like+domain+in+a+selenoprotein,+widespread+in+the+tree+of+life.">A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.</a> PLoS One. 7 (2), e32138 (2012).</li><li>Han, S. J., Lee, B. C., Yim, S. H., Gladyshev, V. N., Lee, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mammalian+selenoprotein+o:+A+redox-active+mitochondrial+protein.">Characterization of mammalian selenoprotein o: A redox-active mitochondrial protein.</a> PLoS One. 9 (4), e95518 (2014).</li><li>Sreelatha, A., 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=Protein+ampylation+by+an+evolutionarily+conserved+pseudokinase.">Protein ampylation by an evolutionarily conserved pseudokinase.</a> Cell. 175 (3), 809-821.e19 (2018).</li><li>Frese, 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+alarmone+diadenosine+tetraphosphate+as+a+cosubstrate+for+protein+ampylation.">The alarmone diadenosine tetraphosphate as a cosubstrate for protein ampylation.</a> Angew Chem Int Ed Engl. 62 (8), e202213279 (2023).</li><li>Mostert, D., 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=Pronucleotide+probes+reveal+a+diverging+specificity+for+ampylation+vs+umpylation+of+human+and+bacterial+nucleotide+transferases.">Pronucleotide probes reveal a diverging specificity for ampylation vs umpylation of human and bacterial nucleotide transferases.</a> Biochemistry. 63 (5), 651-659 (2024).</li><li>Yang, Y., 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+ydiu+domain+modulates+bacterial+stress+signaling+through+Mn2+-dependent+umpylation.">The ydiu domain modulates bacterial stress signaling through Mn2+-dependent umpylation.</a> Cell Rep. 32 (12), 108161 (2020).</li><li>Mukherjee, M., Sreelatha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+selenoprotein+o+substrates+using+a+biotinylated+atp+analog.">Identification of selenoprotein o substrates using a biotinylated atp analog.</a> Methods Enzymol. 662, 275-296 (2022).</li><li>Kroeger, 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=Edta+aggregates+induce+sypro+orange-based+fluorescence+in+thermal+shift+assay.">Edta aggregates induce sypro orange-based fluorescence in thermal shift assay.</a> PLoS One. 12 (5), e0177024 (2017).</li><li>Kotov, V., 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=In-depth+interrogation+of+protein+thermal+unfolding+data+with+moltenprot.">In-depth interrogation of protein thermal unfolding data with moltenprot.</a> Protein Sci. 30 (1), 201-217 (2021).</li><li>Wu, T., Gale-Day, Z. J., Gestwicki, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dsfworld:+A+flexible+and+precise+tool+to+analyze+differential+scanning+fluorimetry+data.">Dsfworld: A flexible and precise tool to analyze differential scanning fluorimetry data.</a> Protein Sci. 33 (6), e5022 (2024).</li><li>Reinhard, L., Mayerhofer, H., Geerlof, A., Mueller-Dieckmann, J., Weiss, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+protein+buffer+cocktails+using+thermofluor.">Optimization of protein buffer cocktails using thermofluor.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 69 (Pt 2), 209-214 (2013).</li><li>Ribeiro, A. J. 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=Emerging+concepts+in+pseudoenzyme+classification,+evolution,+and+signaling.">Emerging concepts in pseudoenzyme classification, evolution, and signaling.</a> Sci Signal. 12 (594), eaat9797 (2019).</li><li>Goldberg, T., Sreelatha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+functions+of+pseudoenzymes.">Emerging functions of pseudoenzymes.</a> Biochem J. 480 (10), 715-728 (2023).</li><li>Pon, A., Osinski, A., Sreelatha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+pseudokinases:+A+look+at+the+untapped+enzymatic+potential+of+pseudokinases.">Redefining pseudokinases: A look at the untapped enzymatic potential of pseudokinases.</a> IUBMB Life. 75 (4), 370-376 (2023).</li><li>Murphy, J. 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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