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 decoupled switching mechanism. This arises from its unique off- (non-fluorescent) state which is a UV absorbing hydrated form of the typical green (G)FP chromophore. Here we report ultrafast dynamics of the off- to on-state transition by complementary ultrafast optical and vibrational transient absorption. The ultrafast photodehydration observed affords the opportunity to probe the response of a protein to bond breaking and forming events in real time. The excited electronic state of the chromophore undergoes photodehydration with a yield estimated at several per-cent in a non-single exponential fashion in tens to hundreds of picoseconds. The primary product formed is the cis protonated form of the GFP chromophore in a perturbed H-bonded environment. This primary product relaxes on a few microsecond timescale by a mechanism suggested to involve changes to a glutamic acid residue and modifications of the amide backbone, possibly involving a carbonyl to imine reaction. The final deprotonation of the chromophore required for on-state formation was not observed, so probably occurs in > 1 ms. 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 its photoswitching properties.