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Kaede, an analogue of green fluorescent protein (GFP), is a green-to-red photoconvertible fluorescent protein used as an in vivo ‘optical highlighter’ in bioimaging. The fluorescence quantum yield of the red Kaede protein is lower than that of GFP, suggesting that increasing the conjugation modifies the electronic relaxation pathway. Using a combination of anion photoelectron spectroscopy and electronic structure calculations, we find that the isolated red Kaede protein chromophore in the gas phase is deprotonated at the imidazole ring, unlike the GFP chromophore that is deprotonated at the phenol ring. We find evidence of an efficient electronic relaxation pathway from higher lying electronically excited states to the S1 state of the red Kaede chromophore that is not accessible in the GFP chromophore. Rapid autodetachment from high lying vibrational states of S1 is found to compete efficiently with internal conversion to the ground electronic state.

BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered.

The S 2 fluorescence of malachite green is measured with sub 100 fs time resolution. Ultrafast spectral dynamics in the S 2 state preceding S 2 decay are resolved. Measurements in different solvents show that these sub 100 fs dynamics are insensitive to medium polarity and viscosity. They are thus assigned to ultrafast structural evolution between the S 2 Franck–Condon and equilibrium configurations.

Fluorescent proteins exhibit a very diverse range of photochemical behaviour, from efficient fluorescence through photochromism to photochemical reactivity. Remarkably this diverse behaviour arises from chromophores which have very similar structures. Here we describe measurements and modelling of the excited state dynamics in the chromophores of GFP (HBDI) and the kindling fluorescent protein, KFP (FHBMI). The methods are ultrafast fluorescence spectroscopy with sub 50 fs time resolution and the modelling is based on the Smoluchowski equation. The excited state decays of both chromophores are very fast, longer for their anions than for the neutral form and independent of wavelength. Detailed studies show the mean fluorescence wavelength to be independent of time. The excited state decay times are also observed to be a very weak function of solvent polarity and viscosity. These results are modelled utilising recently calculated potential energy surfaces for the ground and excited states as a function of the twist coordinates about the two bridging bonds of the chromophore. For FHBMI and the scarce data on the neutral HBDI the calculations are not successful suggesting the need for refinement of these potential energy surfaces. For HBDI in methanol the simulation is successful provided a strong dependence of the radiationless decay rate on the coordinate is assumed. Such dependence should be included in future calculations of excited state dynamics. When the simulations are extended to more viscous solvents they fail to reproduce the observed weak viscosity dependence. The implications of these results for the nature of the coordinate leading to radiationless decay in the chromophore and for the photodynamics of fluorescent proteins are discussed.

Light-driven molecular motors convert light into mechanical energy via excited state reactions. In this work we follow sub-picosecond primary events in the cycle of a two-stroke unidirectional motor by fluorescence up-conversion and transient absorption.