<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

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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

649 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. ...

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 ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

生物工程

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

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 ...

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

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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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

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

利用高含量成像对增强细胞中的目标参与进行量化

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

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

使用修饰的合成寡核苷酸检测核酸代谢酶

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

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

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

利用高含量成像对增强细胞中的目标参与进行量化

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

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

使用修饰的合成寡核苷酸检测核酸代谢酶

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