Differential Scanning Fluorimetry (DSF) Methods for Studying Protein Stability
研究蛋白质稳定性的差示扫描荧光 (DSF) 方法
基本信息
- 批准号:10184149
- 负责人:
- 金额:$ 39.05万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-08-05 至 2025-05-31
- 项目状态:未结题
- 来源:
- 关键词:AlgorithmsAlzheimer&aposs DiseaseBenchmarkingBindingBinding ProteinsCD69 antigenChemicalsChemistryChromatin Remodeling FactorCircular DichroismClinicalCollaborationsComplexCystic FibrosisDataData AnalysesDatabasesDefectDifferential Scanning CalorimetryDiseaseDrug TargetingDyesEquationFailureFluorescenceGoalsGoldHydrophobicityIndividualKineticsKnowledgeLabelLibrariesLigand BindingLinkMachine LearningMeasurementMeasuresMembraneMethodsModalityMolecular ConformationMonitorMultiprotein ComplexesNeurodegenerative DisordersNucleic Acid BindingOrangesPharmaceutical ChemistryPilot ProjectsProcessPropertyProteinsReportingResearchRoleScanningSeriesSpecificityStructureStructure-Activity RelationshipSystemTechnologyTemperatureTestingTherapeuticTimeWorkaerobic respiration control proteinbasecheminformaticsconformational conversiondesignenzyme activityexperimental studyhigh throughput screeninghigh throughput technologyhuman diseaseimprovedinnovationinstrumentinterestlarge datasetsmeltingmetalloenzymenew technologynext generationnovel therapeuticsprotein complexprotein foldingprotein misfoldingreconstitutionsimulationsmall moleculesuccesstau Proteinstheoriesweb portal
项目摘要
Abstract. There is great interest in technologies that measure protein stability, because many devastating
diseases (e.g. cystic fibrosis, Alzheimer’s disease) are linked to protein misfolding and instability. One especially
promising way to treat these diseases is to use small molecules, termed “correctors” that bind to the damaged
protein and partially restore its folding. Multiple correctors have received FDA approval (e.g. ivacaftor, tafamadis,
migalastat), but there are hundreds of additional misfolding diseases. What are the hurdles to the rapid discovery
of additional correctors? One important barrier is that previous correctors have been uncovered through
prolonged searches, using specialized (i.e. target-specific) technologies that are not versatile enough for use
across many proteins-of-interest (POIs). Here, we propose next-generation Differential Scanning Fluorimetry
(DSF) to fill this gap. In a typical DSF experiment, a POI is heated in a qPCR instrument and its un-folding is
monitored by its binding to a solvatochromatic dye (e.g. Sypro Orange, SO). The resulting temperature vs.
fluorescence curves are then used to estimate the melting transition (Tm), with putative correctors identified by
their effect on this value (DTm). DSF is versatile because it does not require protein labeling or structural
knowledge. Moreover, unlike comparable platforms, such as circular dichroism (CD) or differential scanning
calorimetry (DSC), DSF is amenable to 384-well plate format, facilitating large-scale chemical screens. While
DSF has the potential to transform corrector discovery, there are major hurdles to overcome. For example, DSF
often fails because SO does not bind the target protein or it binds to hydrophobic patches on the native state,
obscuring the Tm. Further, for some POIs, the temperature-fluorescence curves are complex, with multiple
transitions, and therefore not readily analyzed or fit using standard equations. Based on our preliminary screens
of ~50 different proteins, these issues cause DSF to fail in more than 60% of cases. We propose to solve these
issues through disruptive innovations: (SA1) Design and synthesis of next-generation dye libraries that
significantly expand the scope of DSF and (SA2) Theory- and experiment-driven, dramatic improvements in data
analysis, enabled by machine learning and made publicly available through a web portal (DSFWorld).
Encouraged by preliminary success, we also propose to: (SA3) Expand the scope of DSF applications by
pioneering studies of multi-protein complexes and conformational changes. Importantly, we will benchmark each
of these innovations against current state-of-the-art approaches, with a focus on a critical understanding of
strengths and weaknesses. Together, these studies are expected to dramatically expand the scope of DSF
technology.
摘要。人们对测量蛋白质稳定性的技术非常感兴趣,因为许多技术具有破坏性
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
Jason E Gestwicki其他文献
Exploration of the Binding Determinants of Protein Phosphatase 5 (PP5) Reveals a Chaperone-Independent Activation Mechanism.
蛋白磷酸酶 5 (PP5) 结合决定因素的探索揭示了一种不依赖分子伴侣的激活机制。
- DOI:
- 发表时间:
2024 - 期刊:
- 影响因子:4.8
- 作者:
Shweta Devi;Annemarie Charvat;Zoe Millbern;Nelson Vinueza;Jason E Gestwicki - 通讯作者:
Jason E Gestwicki
Jason E Gestwicki的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('Jason E Gestwicki', 18)}}的其他基金
Chemical Biology Approaches to Studying Collagen IV Stability
研究胶原蛋白 IV 稳定性的化学生物学方法
- 批准号:
10723042 - 财政年份:2023
- 资助金额:
$ 39.05万 - 项目类别:
Research Training in Chemistry and Chemical Biology
化学和化学生物学研究培训
- 批准号:
10410908 - 财政年份:2022
- 资助金额:
$ 39.05万 - 项目类别:
Research Training in Chemistry and Chemical Biology
化学和化学生物学研究培训
- 批准号:
10624303 - 财政年份:2022
- 资助金额:
$ 39.05万 - 项目类别:
Differential Scanning Fluorimetry (DSF) Methods for Studying Protein Stability
研究蛋白质稳定性的差示扫描荧光 (DSF) 方法
- 批准号:
10626847 - 财政年份:2021
- 资助金额:
$ 39.05万 - 项目类别:
Differential Scanning Fluorimetry (DSF) Methods for Studying Protein Stability
研究蛋白质稳定性的差示扫描荧光 (DSF) 方法
- 批准号:
10462611 - 财政年份:2021
- 资助金额:
$ 39.05万 - 项目类别:
Activation of the 20S Proteasome to Normalize Tau Homeostasis
激活 20S 蛋白酶体使 Tau 稳态正常化
- 批准号:
9329344 - 财政年份:2016
- 资助金额:
$ 39.05万 - 项目类别:
Chemical Probes and Chaperone-Accelerated Turnover of Tau
化学探针和分子伴侣加速 Tau 蛋白的周转
- 批准号:
8519207 - 财政年份:2012
- 资助金额:
$ 39.05万 - 项目类别:
Natural Product-Inspired Method for Enhancing HIV Protease Inhibitors
增强 HIV 蛋白酶抑制剂的天然产物方法
- 批准号:
8259867 - 财政年份:2012
- 资助金额:
$ 39.05万 - 项目类别:
Natural Product-Inspired Method for Enhancing HIV Protease Inhibitors
增强 HIV 蛋白酶抑制剂的天然产物方法
- 批准号:
8416319 - 财政年份:2012
- 资助金额:
$ 39.05万 - 项目类别: