Elucidating photocycle in large Stokes shift red fluorescent proteins: Focus on mKeima

Abstract

AbstractLarge Stokes shift red fluorescent proteins (LSS‐RFPs) are genetically encoded and exhibit a significant difference of a few hundreds of nanometers between their excitation and emission peak maxima (i.e., the Stokes shift). These LSS‐RFPs (absorbing blue light and emitting red light) feature a unique photocycle responsible for their significant Stokes shift. The photocycle associated with this LSS characteristic in certain RFPs is quite perplexing, hinting at the complex nature of excited‐state photophysics. This article provides a brief review on the fundamental mechanisms governing the photocycle of various LSS‐RFPs, followed by a discussion on experimental results on mKeima emphasizing its relaxation pathways which garnered attention due to its >200 nm Stokes shift. Corroborating steady‐state spectroscopy with computational studies, four different forms of chromophore of mKeima contributing to the cis‐trans conformers of the neutral and anionic forms were identified in a recent study. Furthering these findings, in this account a detailed discussion on the photocycle of mKeima, which encompasses sequential excited‐state isomerization, proton transfer, and subsequent structural reorganization involving three isomers, leading to an intriguing temperature and pH‐dependent dual fluorescence, is explored using broadband femtosecond transient absorption spectroscopy.