Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O
Summary
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.
Abstract
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.
Introduction
Much of the human proteome remains poorly characterized. The "streetlight effect" 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's applications. The following protocol can be readily adapted to optimize and examine the interactions of other proteins and additives of interest.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Adenosine 5′-triphosphate disodium salt hydrate | Sigma Aldrich | A2383-10G | used for representative results but not required to perfom TSA |
| Avanti J-15R with microplate carrier assembly | Beckman Coulter | C19416 | |
| CFX Opus 384 Real-time PCR system | Bio-Rad | 12011452 | |
| Hard-Shell 384-Well PCR Plates, thin wall, skirted, clear/white | Bio-Rad | HSP3805 | |
| Magnesium Chloride | Sigma Aldrich | M8266-100G | used for representative results but not required to perfom TSA |
| Manganese (II) chloride tetrahydrate | Sigma Aldrich | M3634-500G | used for representative results but not required to perfom TSA |
| Microseal 'B' PCR Plate Sealing Film, adhesive, optical | Bio-Rad | MSB1001 | |
| SYPRO Orange Protein Gel stain | Sigma Aldrich | S5692-500UL | |
| Uridine 5′-triphosphate trisodium salt hydrate | Sigma Aldrich | U6625-100MG | used for representative results but not required to perfom TSA |
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