Name: utilizing thermal shift assay to probe substrate binding to selenoprotein o
Uploaded: 2024-08-09T14:45:00.000+00:00
Duration: 3 min 9 s
Description: Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although ...

<meta charset="utf8"></head><body>用于蛋白质-配体相互作用的高通量热位移检测

<ol>
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<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

<meta charset="utf8"></head><body>作者聚焦：通过热位移分析推进蛋白质的结构和生化研究

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

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

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

526 次观看.  University of Texas Southwestern Medical Center. 我们旨在研究功能未知的蛋白质的生化活性。辅因子结合不仅对某些蛋白质的活性至关重要，而且还可能改变它们的热稳定性。这种现象的一个实际应用在于利用蛋白质熔解温度测量的热稳定性变化来分析配体结合。热位移分析是一种多功能工具，用于研究蛋白质与潜在辅助因子或药物的相互作用，以及确定晶体学的稳定条件。与其他筛选方法...

视频: 作者聚焦：通过热位移分析推进蛋白质的结构和生化研究

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.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

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

科研

生物化学

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.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

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.

寡肽竞争测定磷酸化位点测定

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.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

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.

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

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

遗传学

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.

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

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.

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

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

癌症研究

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.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

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.

孔雀石绿测定法用于发现热休克蛋白 90 抑制剂

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

使用合成寡核苷酸的 DNA 酶检测

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.

作者聚焦：表征来自极端微生物和常见病原体的新型酶以了解 DNA 修复和复制

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.

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

副本

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此视频中的章节

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

寡肽竞争测定磷酸化位点测定

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

通过 SHP2 （PTPN11）磷酸酶抑制剂评估细胞目标参与度

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

孔雀石绿测定法用于发现热休克蛋白 90 抑制剂

使用修饰的合成寡核苷酸检测核酸代谢酶

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

副本

摘要

探索更多视频

此视频中的章节

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

寡肽竞争测定磷酸化位点测定

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

孔雀石绿测定法用于发现热休克蛋白 90 抑制剂

使用修饰的合成寡核苷酸检测核酸代谢酶

通过 SHP2 （PTPN11）磷酸酶抑制剂评估细胞目标参与度