In depth, thermodynamic analysis of Ca2+ binding to human cardiac troponin C: Extracting buffer-independent binding parameters
In depth, thermodynamic analysis of Ca2+ binding to human cardiac troponin C: Extracting buffer-independent binding parameters
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DOI：
10.1016/j.bbapap.2019.01.004
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发表时间：
2019-04-01
影响因子：
3.2
通讯作者：
Spuches, Anne M.
中科院分区：
文献类型：
--
作者：
Johnson, Rachel A.;Fulcher, Lindsay M.;Spuches, Anne M.
背景：表征金属-生物分子相互作用背后的热力学参数是理解金属离子在生物学中所起作用的基础。等温滴定量热法(ITC)是获得这些数据的“黄金标准”。然而，除了金属与蛋白质的结合外，在对金属结合体系进行有意义的比较之前，还必须考虑金属与缓冲液的相互作用等额外的平衡。方法：在本研究中，通过ITC滴定钙离子与三种缓冲液(Bis-Tris，MES和MOPS)的结合热力学。这些数据被用来提取钙离子与人心肌肌钙蛋白C(HcTnC)结合的缓冲区无关参数。hcTnC是一种含EF手性蛋白的心肌收缩所必需的蛋白质。结果：钙离子与hcTnC C-结构域和N-结构域结合释放的质子数分别为1.1和1.2。这些值允许测定不依赖于缓冲液的钙离子-hcTnC结合的热力学参数，提取的数据在被测试的缓冲液中符合得很好。测定了缓冲液和pH调节的钙离子与hcTnC N-结构域结合的参数，发现在水溶液条件下和生理离子强度下的钙离子结合是热力学上有利的，并且是由熵驱动的。结论：缓冲体系之间的数据的一致性以及理论和实验质子释放的相似性表明了所用方法的可靠性和提取金属-缓冲液相互作用在这些研究中的重要性。
Background: Characterizing the thermodynamic parameters behind metal-biomolecule interactions is fundamental to understanding the roles metal ions play in biology. Isothermal Titration Calorimetry (ITC) is a "gold standard" for obtaining these data. However, in addition to metal-protein binding, additional equilibria such as metal-buffer interactions must be taken into consideration prior to making meaningful comparisons between metal-binding systems.Methods: In this study, the thermodynamics of Ca2+ binding to three buffers (Bis-Tris, MES, and MOPS) were obtained from Ca2+-EDTA titrations using ITC. These data were used to extract buffer-independent parameters for Ca2+ binding to human cardiac troponin C (hcTnC), an EF-hand containing protein required for heart muscle contraction.Results: The number of protons released upon Ca2+ binding to the C- and N-domain of hcTnC were found to be 1.1 and 1.2, respectively. These values permitted determination of buffer-independent thermodynamic parameters of Ca2+-hcTnC binding, and the extracted data agreed well among the buffers tested. Both buffer and pH adjusted parameters were determined for Ca2+ binding to the N-domain of hcTnC and revealed that Ca2+ binding under aqueous conditions and physiological ionic strength is both thermodynamically favorable and driven by entropy.Conclusions: Taken together, the consistency of these data between buffer systems and the similarity between theoretical and experimental proton release is indicative of the reliability of the method used and the importance of extracting metal-buffer interactions in these studies.General significance: The experimental approach described herein is clearly applicable to other metal ions and other EF-hand protein systems.