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. 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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, 21-28 (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><tbody><tr><td>Adenosine 5&amp;prime;-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 &#039;B&#039; 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&amp;prime;-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.

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...

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.

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 ...

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

Research

JoVE Journal

Biochemistry

565 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. 

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

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Genetics

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

Cancer Research

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is dependent on its ability to hydrolyze adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and free phosphate. The ATPase activity of Hsp90 is linked to its chaperoning function; ATP binds to the N-terminal domain of the Hsp90, and disrupting its binding was found to be the most successful strategy in suppressing Hsp90 function. The ATPase activity can be measured by a colorimetric malachite green assay, which determines the amount of free phosphate formed by ATP hydrolysis. Here, a procedure for determining the ATPase activity of yeast Hsp90 by using the malachite green phosphate assay kit is described. Further, detailed instructions for the discovery of Hsp90 inhibitors by taking geldanamycin as an authentic inhibitor is provided. Finally, the application of this assay protocol through the high-throughput screening (HTS) of inhibitor molecules against yeast Hsp90 is discussed.

The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is ...

Assays for DNA Enzymes Using Synthetic Oligonucleotides

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Summary

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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

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