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.