Probing a Polar Cluster in the Retinal Binding Pocket of Bacteriorhodopsin by a Chemical Design Approach

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C13 of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.

细菌视紫红质（bacteriorhodopsin）的视黄醛（retinal）分子周围环绕着极性氨基酸簇，该结构负责捕获光能以驱动质子泵过程。基于既往研究成果，我们已证实苏氨酸（threonine）90是该极性氨基酸簇中兼具功能与结构核心作用的关键氨基酸。为实现表型挽救，我们通过化学手段设计了一种视黄醛类似物，以补偿细菌视紫红质T90A突变所引发的显著效应。该类似物将视黄醛碳氢链C13位的甲基替换为乙基，即20-甲基视黄醛。我们分析了将野生型与T90A突变脱辅基蛋白（apoprotein）分别与全反式视黄醛（all-trans-retinal）及其20-甲基衍生物（后文简称13-乙基视黄醛）进行重构后的效果。生物物理表征结果显示，恢复90号残基与视黄醛之间的空间相互作用，可优化生色团（chromophore）的结合环境，但不足以实现完全的表型挽救。对这些经化学工程改造的发色蛋白的表征研究，进一步揭示了氢键网络以及细菌视紫红质与其他微生物感官视紫红质（microbial sensory rhodopsins）中视黄醛结合口袋相关的空间相互作用所发挥的作用。