Soon after Osamu Shimomura discovered green fluorescent protein (GFP) from jellyfish, <i>Aequorea Victoria</i>, different colored fluorescent proteins (FPs) were identified from non-bioluminescent Anthozoa homologs of GFP and produced through various mutations. Apart from excited state proton transfer (ESPT), several other photoinduced reactions play an important role in the origin of colors in FPs which include photooxidation, photoreduction, photoisomerization, making & breaking of the covalent bond which further results in various interesting phenomena like large-stoke shifts, phototoxicity, photoactivation, photoconversion, photo-switching, the production of long-lived intermediates, blinking, permanent bleaching.<br/>The chromophore of red fluorescent proteins (RFPs) has an extended N-acylimine group comparative to GFP chromophore which principally increases the conjugation and thus fluoresces in higher wavelength regions. Two ground state forms of Wild-type GFP and mKeima (a variant of RFP) have been known: neutral form and anionic form. The neutral form after excitation, undergoes an ESPT on a timescale of a few picoseconds, yielding the anionic form in the excited state which then fluoresces, and finally returns to neutral form via proton back-transfer in the ground state, termed as ground state proton transfer (GSPT). In this work, we have investigated the driving force behind the unusual phenomena of “reverse protonation” in mKeima in the different local environments by changing the pH. In addition, conformational heterogeneity of the chromophore showing reverse protonation effect in mKeima is reported in different pH environments. The role of the local environment of protein on the ultrafast excited-state dynamics coupled with the conformational changes in multiple species of chromophore following reverse protonation is explored. We further investigated the origin of dual-fluorescence in mKeima at low pH conditions and utilized this unique property in dual-color bioimaging.

Unravelling the mechanism of reverse protonation in mKeima: Multiple conformers & their ultrafast excited-state dynamics

ACS Spring 2022

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