Department of Biomedical Engineering and Genome Center, 451 East Health Science Drive, University of California , Davis, California 95616-8816, United States.
Biochemistry. 2014 Apr 15;53(14):2380-90. doi: 10.1021/bi4013808. Epub 2014 Apr 2.
The bacteriorhodopsin (BR) Asp96Gly/Phe171Cys/Phe219Leu triple mutant has been shown to translocate protons 66% as efficiently as the wild-type protein. Light-dependent ATP synthesis in haloarchaeal cells expressing the triple mutant is 85% that of the wild-type BR expressing cells. Therefore, the functional activity of BR seems to be largely preserved in the triple mutant despite the observations that its ground-state structure resembles that of the wild-type M state (i.e., the so-called cytoplasmically open state) and that the mutant shows no significant structural changes during its photocycle, in sharp contrast to what occurs in the wild-type protein in which a large structural opening and closing occurs on the cytoplasmic side. To resolve the contradiction between the apparent functional robustness of the triple mutant and the presumed importance of the opening and closing that occurs in the wild-type protein, we conducted additional experiments to compare the behavior of wild-type and mutant proteins under different operational loads. Specifically, we characterized the ability of the two proteins to generate light-driven proton currents against a range of membrane potentials. The wild-type protein showed maximal conductance between -150 and -50 mV, whereas the mutant showed maximal conductance at membrane potentials >+50 mV. Molecular dynamics (MD) simulations of the triple mutant were also conducted to characterize structural changes in the protein and in solvent accessibility that might help to functionally contextualize the current-voltage data. These simulations revealed that the cytoplasmic half-channel of the triple mutant is constitutively open and dynamically exchanges water with the bulk. Collectively, the data and simulations help to explain why this mutant BR does not mediate photosynthetic growth of haloarchaeal cells, and they suggest that the structural closing observed in the wild-type protein likely plays a key role in minimizing substrate back flow in the face of electrochemical driving forces present at physiological membrane potentials.
菌紫质（BR）Asp96Gly/Phe171Cys/Phe219Leu 三重突变体已被证明能够有效地转运质子，效率为野生型蛋白的 66%。在表达三重突变体的盐杆菌细胞中，光依赖性 ATP 合成是野生型 BR 表达细胞的 85%。因此，尽管观察到其基态结构类似于野生型 M 态（即所谓的细胞质开放态），并且突变体在其光循环过程中没有显示出明显的结构变化，但 BR 的功能活性在三重突变体中似乎基本得到保留，这与野生型蛋白形成鲜明对比，在野生型蛋白中，细胞质侧会发生较大的结构开合。为了解决三重突变体的明显功能稳健性与野生型蛋白中发生的开合作用的假定重要性之间的矛盾，我们进行了额外的实验来比较野生型和突变型蛋白在不同操作负载下的行为。具体来说，我们研究了两种蛋白在一系列膜电位下产生光驱动质子电流的能力。野生型蛋白在-150 至-50 mV 的膜电位下显示出最大电导率，而突变体则在> +50 mV 的膜电位下显示出最大电导率。还对三重突变体进行了分子动力学（MD）模拟，以表征蛋白和溶剂可及性的结构变化，这可能有助于从功能上解释电流-电压数据。这些模拟表明，三重突变体的细胞质半通道是持续开放的，并与主体进行动态水交换。总的来说，这些数据和模拟有助于解释为什么这种突变体 BR 不能介导盐杆菌细胞的光合作用生长，并且它们表明在生理膜电位下存在电化学驱动力时，观察到的野生型蛋白的结构关闭可能在最小化底物回流方面发挥关键作用。
