Aspartate/alanine exchange transporter (AspT) is a secondary transporter isolated from the lactic acid bacterium Tetragenococcus halophilus D10 strain. This transporter cooperates with aspartate decarboxylase to produce proton-motive force through decarboxylative phosphorylation. A method that successfully analyzes the AspT mechanism could serve as a prototype for elucidating the substrate transport mechanism of other exchange transporters; therefore, the purpose of this study was to search for conditions that improve the thermal stability of AspT for 3D structure analysis. We used the fluorescence size-exclusion chromatography–based thermostability assay to evaluate conditions that contribute to AspT stability. We found that the AspT thermostability was enhanced at pH 5.0–6.0 and in the presence of Na+ and Li+. Pyridoxal phosphate, a coenzyme of aspartate decarboxylase, also had a thermostabilizing effect on AspT. Under the conditions obtained from these results, it was possible to increase the temperature at which 50% of dimer AspT remained by 14°C. We expect these conditions to provide useful information for future structural analysis of AspT.

中文翻译：

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嗜盐四联球菌天冬氨酸/丙氨酸交换转运蛋白的热稳定性优化

天冬氨酸/丙氨酸交换转运蛋白 (AspT) 是从乳酸菌嗜盐四联球菌 D10 菌株中分离出来的二级转运蛋白。该转运蛋白与天冬氨酸脱羧酶配合，通过脱羧磷酸化产生质子动力。成功分析 AspT 机制的方法可以作为阐明其他交换转运蛋白的底物转运机制的原型；因此，本研究的目的是寻找提高 AspT 热稳定性的条件以进行 3D 结构分析。我们使用基于荧光尺寸排阻色谱的热稳定性测定来评估有助于 AspT 稳定性的条件。我们发现 AspT 的热稳定性在 pH 5.0-6.0 以及 Na+ 和 Li+ 存在的情况下增强。磷酸吡哆醛是天冬氨酸脱羧酶的辅酶，对 AspT 也具有热稳定作用。在由这些结果获得的条件下，可以将二聚体AspT残留50％的温度提高14℃。我们希望这些条件能为 AspT 的未来结构分析提供有用的信息。