Unveiling the Diverse Light-Driven Functions of Microbial Rhodopsins through Integrated Mechanistic Studies

Microbial rhodopsins are light-responsive membrane proteins using a retinal chromophore widely distributed among diverse microorganisms. Over the past years, we have discovered a number of microbial rhodopsins with unprecedented molecular functions. To achieve a comprehensive understanding of these proteins, we have combined various time-resolved and vibrational spectroscopic methods with three-dimensional structural information obtained by X-ray crystallography and cryo-electron microscopy, as well as insights from theoretical calculations. Through these integrated approaches, we have explored in detail how the small structural change of retinal isomerization is coupled to large-scale protein conformational changes and ultimately to biological functions. These studies revealed that the diverse functions of microbial rhodopsins, including light-driven ion pump, light-gated ion channel, light signal transduction, and regulation of gene expression or enzymatic activity, arise from differences in a series of proton transfer reactions. We further demonstrated that molecular function is largely determined by the combination pattern of three specific residues, termed the functional motif. By focusing on genes with previously uncharacterized motifs, we discovered the first outward Na+-pumping and inward H+-pumping rhodopsins, and have reported more than ten new rhodopsin families that are distinct from previously known groups. In addition, we developed machine-learning models capable of predicting absorption wavelengths solely from amino acid sequences. Using this model, we identified more than 30 long-wavelength-absorbing rhodopsins. In this article, we provide an overview of our studies on microbial rhodopsins, from the discovery of novel molecular functions to the elucidation of their mechanisms.