Mapping the Complete Photocycle that Powers a Large Stokes Shift Red Fluorescent Protein

Abstract

Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexisting cis-and trans-isomers, holds significance as an archetypal system for LSS emission due to excited-state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time-resolved electronic spectroscopies, aided by quantum calculations, to dissect the cisand trans-mKeima photocycle from ESPT, isomerization, to ground-state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states.Importantly, the deprotonated trans-isomer governs LSS emission at 620 nm, while the deprotonated cis-isomer's 520 nm emission is weak due to an ultrafast cis-to-trans isomerization. Complementary spectroscopic techniques as a table-top toolset are thus essential to study photochemistry in physiological environments.

Unveiling the Role of Hidden Isomers in Large Stokes Shift in mKeima: Harnessing pH-Sensitive Dual-Emission in Bioimaging

“…Preliminary results of the studies presented here reported possible involvement of different forms in the photocycle of mKeima for the first time (communicated elsewhere). Quite recently, Wang et al reported a similar study considering a model involving concurrent photocycles for both cis and trans forms, where the cis/trans anionic form was identified as the minor/major emissive species and a slow (120 ps) isomerization of the cis anionic form in the excited state to the trans anionic form in the ground state is proposed . However, this contradicts the inference drawn from pH-dependent absorption/crystallographic studies that the cis anionic form is the major emissive species (and also the fact that the emissive form of most RFPs is the cis form − ).…”

“…Quite recently, Wang et al reported a similar study considering a model involving concurrent photocycles for both cis and trans forms, where the cis/trans anionic form was identified as the minor/major emissive species and a slow (120 ps) isomerization of the cis anionic form in the excited state to the trans anionic form in the ground state is proposed. 56 However, this contradicts the inference drawn from pH-dependent absorption/crystallographic studies that the cis anionic form is the major emissive species 24 (and also the fact that the emissive form of most RFPs is the cis form 28−32 ). Further, as evidenced from spectral decomposition as well as crystallographic studies, 24 the cis neutral form being a minor form having negligible absorption at 400 nm is unlikely to participate in the initial steps of the photocycle.…”

“…Further, as evidenced from spectral decomposition as well as crystallographic studies, 24 the cis neutral form being a minor form having negligible absorption at 400 nm is unlikely to participate in the initial steps of the photocycle. Even if both photocycles involving cis and trans isomers, leading to minor (520 nm) and major (620 nm) emissions, respectively, occur in parallel, 56 both emissions are expected to be enhanced at 77 K; contrary to this, significant enhancement of only the 520 nm emission at 77 K is observed, suggestive of the minor and major emissive forms (cis neutral and cis anionic, respectively) being dynamically connected (through ESPT, which is suppressed at 77 K).…”