生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (4): 11-.doi: 10.3969/j.issn.2095-1736.2023.04.011

• 研究报告 • 上一篇    下一篇

亮氨酸94在光驱质子泵古紫质-4中的功能

刘冬雪， 赵　欣   

  1. 华东师范大学 物理与电子科学学院 上海市磁共振重点实验室， 上海 200241
  • 出版日期:2023-08-18 发布日期:2023-08-10
  • 通讯作者: 赵欣，博士，教授，研究方向为生物核磁共振，E-mail:xzhao@phy.ecnu.edu.cn
  • 作者简介:刘冬雪，硕士，研究方向为生物核磁共振，E-mail:807507277@qq.com
  • 基金资助:
    上海市自然科学基金资助项目(19ZR1416300)； 国家自然科学基金资助项目(21475045, 22074040)

Function analysis of leucine 94 in light-driven proton pump archaerhodopsin-4

LIU Dongxue, ZHAO Xin   

  1. Shanghai Key Laboratory of Magnetic Resonance, School of Physics and Electronic Science, East China Normal University, Shanghai 200241, China
  • Online:2023-08-18 Published:2023-08-10

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摘要： 利用基因定点突变技术分别将bR/aR4（细菌视紫红质/古紫质-4）中L93/L94（亮氨酸93/亮氨酸94）突变为侧链不同的丙氨酸、苯丙氨酸和极性不同的苏氨酸，得到bR、含菌红素BI-aR4和缺失菌红素BE-aR4体系突变蛋白，采用紫外-可见光吸收光谱、闪光光解光谱技术和pH滴定等方法，对比研究L94在aR4中对视黄醛构象、光循环及质子传输的影响及其同菌红素的关系。结果表明，aR4中L94同样会通过空间位阻效应调控视黄醛键合区构象和质子通道的螺旋分布，以维持视黄醛顺-反异构平衡和再异构化过程，并且促进质子的释放，保证蛋白高效的质子泵活性。此外，L94对aR4结构和功能的影响会受到菌红素的调制，在缺失菌红素的情况下L94突变对aR4螺旋的扰动更加剧烈，导致质子供体和质子释放簇周围的氢键网络发生变化。

关键词: 古紫质-4, 光驱质子泵, 亮氨酸94, 菌红素, 细菌视紫红质

Abstract: Site-directed mutation technique was used to mutate L93/L94 (leucine 93/leucine 94) in bR/aR4 (bacteriorhodopsin/archearhodopsin-4) to alanine and phenylalanine with different side chains, and threonine with different polarities, respectively. The corresponding bR, BI-aR4 (bacterioruberin-included), and BE-aR4 (bacterioruberin-excluded) mutants were obtained. UV-visible absorption spectroscopy, flash photolysis spectroscopy, and pH titration measurements were used to study the effects of L94 on the retinal, photocycle, proton pumping, and the relation between L94 and bacterioruberin. The results showed that L94 regulated the conformation and dynamic equilibrium of the retinal and the helical distribution of the proton channel through the steric hindrance in aR4 to maintain the retinal cis-trans isomerization and re-isomerization and promote proton release. In addition, L94 seemed to be modulated by bacterioruberin. The L94 mutants perturb helixes more strongly in BE-aR4, resulting in changing the hydrogen bond network of the proton donor and proton release cluster.

Key words: archearhodopsin-4, light-driven proton pump, leucine 94, bacterioruberin, bacteriorhodopsin

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