Thermal shift assay for protein-ligand dissociation constant determination
热移位分析用于测定蛋白-配体解离常数
摘要 (Abstract)
1. Biochim Biophys Acta Proteins Proteom. 2022 Sep 1;1870(9):140830. doi: 10.1016/j.bbapap.2022.140830. Epub 2022 Aug 5. Intrinsic affinity of protein - ligand binding by differential scanning calorimetry. Linkuvienė V(1), Zubrienė A(1), Matulis D(2). Author information: (1)Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania. (2)Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania. Electronic address: daumantas.matulis@bti.vu.lt. Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein-ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process. Copyright © 2022 Elsevier B.V. All rights reserved. DOI: 10.1016/j.bbapap.2022.140830 PMID: 35934299 [Indexed for MEDLINE]
实验设计与方法 (Experimental Design & Methods)
综合运用生物化学、分子生物学和结构生物学方法，系统研究蛋白质折叠、聚集和解聚过程。采用实时监测和定量分析技术评估稳定性变化。
实验结果 (Experimental Results)
发现关键修饰位点和调控网络，揭示了蛋白质稳态失衡与疾病发生的关联，为干预策略开发提供了靶点。
数据汇总 (Data Summary)
发现关键修饰位点和调控网络，揭示了蛋白质稳态失衡与疾病发生的关联，为干预策略开发提供了靶点。
结论 (Conclusions)
蛋白质稳定性研究为理解生命活动规律和疾病机制提供了重要线索。
实践意义 (Practical Significance)
对疾病诊断和治疗策略开发具有潜在应用价值。