The stabilities of Ca²⁺-regulated ctenophore and coelenterate apo-photoproteins, apo-mnemiopsin (apo-Mne) and apo-aequorin (apo-Aeq), respectively, were compared biochemically, biophysically, and structurally. Despite high degrees of structural and functional conservation, drastic variations in stability and structural dynamics were found between the two proteins. Irreversible thermoinactivation experiments were performed upon incubation of apo-photoproteins at representative temperatures. The inactivation rate constants (k_inact) at 50 °C were determined to be 0.001 and 0.004 min^–1 for apo-Mne and apo-Aeq, respectively. Detailed analysis of the inactivation process suggests that the higher thermostability of apo-Mne is due to the higher activation energy (E_a) and subsequently higher values of ΔH* and ΔG* at a given temperature. According to molecular dynamics simulation studies, the higher hydrogen bond, electrostatic, and van der Waals energies in apo-Mne can validate the relationship between the thermal adaptation of apo-Mne and the energy barrier for the inactivation process. Our results show that favorable residues for protein thermostability such as hydrophobic, charged, and adopted α-helical structure residues are more frequent in the apo-Mne structure. Although the effect of acrylamide on fluorescence quenching suggests that the local flexibility in regions around Trp and Tyr residues of apo-Aeq is higher than that of apo-Mne, which results in it having a better ability to penetrate acrylamide molecules, the root-mean-square fluctuation of helix A in apo-Mne is higher than that in apo-Aeq. It seems that the greater flexibility of apo-Mne in these regions may be considered as a determining factor, affecting the thermal stability of apo-Mne through a balance between structural rigidity and flexibility.

中文翻译：

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栉水母和腔肠动物 Ca2+ 调节的载脂蛋白光蛋白的热稳定性：比较研究

分别从生化、生物物理和结构上比较了 Ca ²⁺调节的栉水母和腔肠动物载脂蛋白、载脂蛋白 (apo-Mne) 和载脂蛋白水母发光蛋白 (apo-Aeq)的稳定性。尽管具有高度的结构和功能保守性，但在两种蛋白质之间发现了稳定性和结构动力学的剧烈变化。在代表性温度下孵育载脂蛋白光蛋白后进行不可逆热灭活实验。50 °C 下的失活速率常数 ( k _inact ) 确定为 0.001 和 0.004 min ^–1分别为 apo-Mne 和 apo-Aeq。失活过程的详细分析表明，apo-Mne 较高的热稳定性是由于较高的活化能 ( E _a ) 和随后较高的 Δ H * 和 Δ G 值* 在给定温度下。根据分子动力学模拟研究，apo-Mne 中较高的氢键、静电和范德华能可以验证 apo-Mne 的热适应与失活过程的能垒之间的关系。我们的结果表明，对蛋白质热稳定性有利的残基，如疏水的、带电的和采用的 α-螺旋结构残基在 apo-Mne 结构中更常见。虽然丙烯酰胺对荧光猝灭的影响表明 apo-Aeq 的 Trp 和 Tyr 残基周围区域的局部灵活性高于 apo-Mne，这导致它具有更好的穿透丙烯酰胺分子的能力，均值根apo-Mne 中螺旋 A 的平方波动高于 apo-Aeq。