Dynamics and Mechanism of Off‐ to On‐Switching in Dreiklang a Decoupled Reversibly Switchable Fluorescent Protein

Dreiklang is a reversibly switchable (rs) fluorescent protein (FP) with a unique off-state, a UV absorbing hydrated form of the typical FP chromophore. Here we report ultrafast dynamics of the off- to on-state transition in Dreiklang using complementary ultrafast optical and vibrational transient absorption to resolve chromophore driven protein structural dynamics. This approach allows observation of the real-time response in a protein to bond breaking and forming events. The excited electronic state decays in a nonsingle exponential fashion in tens to hundreds of picoseconds, undergoing photodehydration with a yield of several per-cent. The primary photoproduct formed is identified as the cis protonated form of the FP chromophore, initially in a perturbed H-bonded environment. This primary product relaxes on a few microseconds timescale by a mechanism involving changes to a glutamic acid residue and modifications of the amide backbone, possibly involving a carbonyl to imine tautomerization. The temporal and spectral resolution of Dreiklang's photodehydration provides data against which to test quantum chemical calculations of reaction dynamics in proteins and suggests a route to modifying and potentially enhancing its photoswitching properties.