A Detailed View on the (Re)isomerization Dynamics in Microbial Rhodopsins Using Complementary Near-UV and IR Readouts

Isomerization is a key process in many (bio)chemical systems. In microbial rhodopsins, the photoinduced isomerization of the all-trans retinal to the 13-cis isomer initiates a cascade of structural changes of the protein. The interplay between these changes and the thermal relaxation of the isomerized retinal is one of the crucial determinants for rhodopsin functionality. It is therefore important to probe this dynamic interplay with chromophore specific markers that combine gapless temporal observation with spectral sensitivity. Here we utilize the near-UV and mid-IR fingerprint region in the framework of a systematic (time-resolved) spectroscopic study on H⁺- (HsBR, (G)PR), Na⁺- (KR2, ErNaR) and Cl^--(NmHR) pumps. We demonstrate that the near-UV region is an excellent probe for retinal configuration and-being sensitive to the electrostatic environment of retinal-even transient ion binding, which allows us to pinpoint protein specific mechanistic nuances and chromophore-charge interactions. The combination of the near-UV and mid-IR fingerprint region hence provides a spectroscopic analysis tool that allows a detailed, precise and temporally fully resolved description of retinal configurations during all stages of the photocycle.