Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
PLoS One. 2022 Dec 1;17(12):e0278417. doi: 10.1371/journal.pone.0278417. eCollection 2022.
The mechanism underlying allostery in hemoglobin (Hb) is still not completely understood. Various models describing the action of allosteric effectors on Hb function have been published in the literature. It has also been reported that some allosteric effectors-such as chloride ions, inositol hexaphosphate, 2,3-diphospho-glycerate and bezafibrate-considerably lower the oxygen affinity of Hb. In this context, an important question is the extent to which these changes influence the conformational dynamics of the protein. Earlier, we elaborated a challenging method based on phosphorescence quenching, which makes characterizing protein-internal dynamics possible in the ms time range. The experimental technique involves phosphorescence lifetime measurements in thermal equilibrium at varied temperatures from 10 K up to 273 K, based on the signal of Zn-protoporphyrin substituted for the heme in the β-subunits of Hb. The thermal activation of protein dynamics was observed by the enhancement of phosphorescence quenching attributed to O2 diffusion. It was shown that the thermal activation of protein matrix dynamics was clearly distinguishable from the dynamic activation of the aqueous solvent, and was therefore highly specific for the protein. In the present work, the same method was used to study the changes in the parameters of the dynamic activation of human HbA induced by binding allosteric effectors. We interpreted the phenomenon as phase transition between two states. The fitting of this model to lifetime data yielded the change of energy and entropy in the activation process and the quenching rate in the dynamically activated state. The fitted parameters were particularly sensitive to the presence of allosteric effectors and could be interpreted in line with results from earlier experimental studies. The results suggest that allosteric effectors are tightly coupled to the dynamics of the whole protein, and thus underline the importance of global dynamics in the regulation of Hb function.
血红蛋白（Hb）变构作用的机制尚不完全清楚。文献中发表了各种描述变构效应物对 Hb 功能作用的模型。据报道，一些变构效应物，如氯离子、肌醇六磷酸、2,3-二磷酸甘油酸和苯扎贝特，可显著降低 Hb 的氧亲和力。在这种情况下，一个重要的问题是这些变化在多大程度上影响蛋白质的构象动力学。早些时候，我们提出了一种基于磷光猝灭的挑战性方法，该方法可以在 ms 时间范围内对蛋白质内部动力学进行特征描述。该实验技术涉及在从 10 K 到 273 K 的不同温度下进行热平衡时的磷光寿命测量，这是基于β亚基中血红素被锌原卟啉取代的信号。通过观察归因于 O2 扩散的磷光猝灭增强，观察到蛋白质动力学的热激活。结果表明，蛋白质基质动力学的热激活与水溶剂的动态激活明显不同，因此对蛋白质具有高度特异性。在本工作中，相同的方法用于研究结合变构效应物诱导的人 HbA 参数变化的蛋白质动态激活。我们将该现象解释为两种状态之间的相变。将该模型拟合到寿命数据中，得到了激活过程中能量和熵的变化以及动态激活状态下的猝灭速率。拟合参数对变构效应物的存在特别敏感，并且可以根据早期实验研究的结果进行解释。结果表明，变构效应物与整个蛋白质的动力学紧密耦合，从而强调了全局动力学在 Hb 功能调节中的重要性。
