Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

Curtis J. Layton and Homme W. Hellinga

首页> 外文期刊>Biochemistry >Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

【24h】

Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

机译：配体诱导的蛋白质热解折叠变化的热力学分析，用于外源荧光染料高通量测定配体亲和力

获取原文

获取原文并翻译 | 示例

掌桥外文数据库（机构版） >>

开具论文收录证明 >>

文献代查 >>

页面导航

摘要
著录项
相似文献
相关主题

摘要

The quantification of protein-ligand interactions is essential for systems biology, drug discovery,nand bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiablensignature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase theirnfluorescence emission intensities upon interaction with the unfolded protein. Thismethod is an experimentallynstraightforward, economical, and high-throughput approach for observing thermal melts using commonlynavailable real-time polymerase chain reaction instrumentation. However, quantitative analysis requiresncareful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model.nWe determine affinity constants by analysis of ligand-mediated shifts inmelting-temperaturemidpoint values.nLigand affinity is determined in a ligand titration series from shifts in free energies of stability at a commonnreference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of thenexperimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinearnbaseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominentnnonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting.We analyze cases innwhich the ligand binds to both the native and denatured state or to the native state only and cases in whichnprotein:ligand stoichiometry needs to treated explicitly.

机译：蛋白质-配体相互作用的定量对于系统生物学，药物发现，生物工程至关重要。配体诱导的蛋白质热稳定性变化提供了一般的，可量化的结合特征，可以用染料如Sypro Orange（SO）进行监测，当与未折叠的蛋白质相互作用时，染料会增加其荧光发射强度。该方法是一种实验上简单，经济，高通量的方法，可使用常用的实时聚合酶链反应仪器观察热熔体。但是，定量分析需要仔细考虑染料介导的报告机制和潜在的热力学模型。n我们通过分析配体介导的转变熔融温度中点值来确定亲和常数.n配体的亲和力是根据配体滴定序列中的自由能变化来确定的。在参考温度下的稳定性。通过将包含变性或线性基线模型的方程式拟合到热变性实验信号的反一阶导数中，可以得到热力学参数。我们应用这些方法来拟合用SO监测的蛋白熔体，该熔体表现出明显的非线性过渡后基线。因此，SO可以扰乱其报告的平衡。我们分析了配体既可以结合天然状态又可以结合变性状态或仅结合天然状态的情况，以及需要明确处理蛋白质：配体化学计量的情况。

著录项

来源
《Biochemistry》 |2010年第51期|p.10831-10841|共11页
作者
Curtis J. Layton and Homme W. Hellinga;
展开▼
作者单位

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, United States;

展开▼
收录信息
原文格式 PDF
正文语种 eng
中图分类
关键词

相似文献

外文文献
中文文献
专利

1. Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes [J] . Layton CJ, Hellinga HW Biochemistry . 2010,第51期

机译：配体诱导的蛋白质热折叠变化的热力学分析，用于高通量测定外源荧光染料配体亲和力
2. Size-Dependent Relationships between Protein Stability and Thermal Unfolding Temperature Have Important Implications for Analysis of Protein Energetics and High-Throughput Assays of Protein-Ligand Interactions [J] . Watson Matthew D., Monroe Jeremy, Raleigh Daniel P. The journal of physical chemistry, B. Condensed matter, materials, surfaces, interfaces & biophysical . 2018,第21期

机译：蛋白质稳定性与热展开温度之间的大小依赖关系对蛋白质能量和蛋白质 - 配体相互作用的高通量测定具有重要意义
3. Ligand-induced thermostability in proteins: Thermodynamic analysis of ANS-albumin interaction [J] . Celej MS, Dassie SA, Freire E, Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics . 2005,第2期

机译：配体诱导的蛋白质热稳定性：ANS-白蛋白相互作用的热力学分析
4. Determination of Site-specific Protein-Ligand Binding Affinities using Protein Ligand Interaction by Mass Spectrometry, Titration, and Hydrogen/Deuterium EXchange (PLMSTEX) Strategy [C] . Jagat Adhikari, Don L. Rempel, Michael L. Gross ASMS Conference on Mass Spectrometry and Allied Topics . 2016

机译：用质谱法测定蛋白质配体，滴定和氢气/氘交换（PLMSTEX）策略的蛋白质配体相互作用测定特异性蛋白质 - 配体结合亲和力
5. Applications of Monte Carlo statistical mechanics to the analysis of protein-ligand binding affinities and to the effects of solvent on reaction rates. [D] . Ostrovsky, Dennis. 2003

机译：蒙特卡洛统计力学在蛋白质-配体结合亲和力分析以及溶剂对反应速率的影响中的应用。
6. Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding [O] . Emma Branigan, Christos Pliotas, Gregor Hagelueken, -1

机译：使用荧光染料和热展开法定量分析膜和可溶性蛋白中的半胱氨酸
7. Size-Dependent Relationships between Protein Stability and Thermal Unfolding Temperature Have Important Implications for Analysis of Protein Energetics and High-Throughput Assays of Protein–Ligand Interactions [O] . Matthew D. Watson, Jeremy Monroe, Daniel P. Raleigh 2017

机译：蛋白质稳定性与热展开温度之间的大小依赖关系对蛋白质能量和蛋白质 - 配体相互作用的高通量测定具有重要意义

Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes

摘要

著录项

相似文献

相关主题

期刊订阅