Hidden photoinduced reactivity of the blue fluorescent protein mKalama1

Abstract

Understanding the photoinduced dynamics of fluorescent proteins is essential for their applications in bioimaging. Despite numerous studies on the ultrafast dynamics, the delayed response of these proteins, which often results in population of kinetically trapped dark states of various origins, is largely unexplored. Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert. This protein shows no excited-state proton transfer during its nanosecond excited-state lifetime; however, its tyrosine-based chromophore undergoes deprotonation coupled to non-radiative electronic relaxation. Such deprotonation causes distinct optical absorption changes in the broad UV-to-NIR spectral range (ca. 300-800 nm); the disappearance of the transient absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of proposed intermediates in the photocycle. We conclude that the non-radiative excited-state decay includes two major branches: internal conversion coupled to intraprotein proton transfer, where a conserved residue E222 serves as the proton acceptor; and ionization induced by two consecutive resonant absorption events, followed by deprotonation of the chromophore radical cation to bulk solvent through a novel water-mediated proton-wire pathway. Our findings open up new perspectives on the dynamics of fluorescent proteins as tracked by its optical transient absorption in the time domain extending up to seconds.

Mechanism of resonant electron emission from the deprotonated GFP chromophore and its biomimetics

“…Upon excitation with a 4 ns 355 nm laser pulse, formation of the solvated electron has indeed been observed in the photocycle of mKalama1, containing the GFP chromophore in the neutral form, enabled through two consecutive resonant absorption events. 41 This protein contains a key V224R substitution in the binding pocket of the chromophore that blocks excited-state protein transfer and immediate redox reactions with E222.…”

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual‐Color Fluorescence Nanoscopy Enabled by a Triplet‐State Mechanism of Primed Conversion

“…Although different transient NIR absorption phenomena have been previously reported for GFP-like FPs, [9] many of the proposed mechanisms fall short of explaining am illisecond-long-lived intermediate state. [5] Specifically,o penshell dianionic chromophore radicals created via photoreduction have been shown to persist for maximally 300-900 ms [9] and cationic chromophore radicals have even shorter lifetimes,a bout 100 ms. [10] We have measured the lifetime of the primed state to be much greater, 5.0 AE 0.2 ms ( Figure 1B), suggesting that neither of the aforementioned mechanisms can serve as an explanation. Notably,i ntersystem crossing (ISC) to the T 1 triplet state has been reported to proceed on longer time scales and could potentially account for this observation.…”

Novel uses of fluorescent proteins

“…Figure 3d shows the TA spectra of selected modulatable FPs in comparison to their DS action spectra. In case of bright blue-light emitting protein omBFP, the DS transient was associated with the anionic species, which are formed as a result of isomerization-coupled internal conversion and deprotonation [67]. The unpublished DS action spectrum of mDsRed had a maximum around 710 nm (see Fig.…”