Search for: All records

Abstract 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. 

Kohinoor is a positive reversibly switching fluorescent protein. Ultrafast electronic and vibrational spectroscopy suggest that the chromophore switching mechanism is steered by dynamics in surrounding protein residues. 

Blue light absorbing flavoproteins play important roles in a variety of photobiological processes. Consequently, there have been numerous investigations of their excited state structure and dynamics, in particular by time-resolved vibrational spectroscopy. The isoalloxazine chromophore of the flavoprotein cofactors has been studied in detail by time-resolved Raman, lending it a benchmark status for mode assignments in excited electronic states of large molecules. However, detailed comparisons of calculated and measured spectra have proven challenging, as there are many more modes calculated than are observed, and the role of resonance enhancement is difficult to characterize in excited electronic states. Here we employ a recently developed approach due to Elles and co-workers ( J. Phys. Chem. A 2018, 122, 8308−8319) for the calculation of resonance-enhanced Raman spectra of excited states and apply it to the lowest singlet and triplet excited states of the isoalloxazine chromophore. There is generally good agreement between calculated and observed enhancements, which allows assignment of vibrational bands of the flavoprotein cofactors to be refined. However, some prominently enhanced bands are found to be absent from the calculations, suggesting the need for further development of the theory. 

The flavin cofactor performs many functions in the cell based on the ability of the isoalloxazine ring to undergo one- or two-electron reduction and form covalent adducts with reactants such as amino acids. In addition, the strong visible absorption of the cofactor is also the basis for flavin-dependent photoreceptors. Vibrational spectroscopy is uniquely suited to studying the mechanism of flavoproteins since the frequency of the vibrational modes is very sensitive to the electronic structure and environment of the isoalloxazine ring. This chapter describes the mechanistic information that can be gained using vibrational spectroscopy as well experimental challenges and approaches that are used to obtain and interpret the complex data contained in a vibrational spectrum.