The photophysics of fluorescent protein chromophores

Fluorescent proteins of the Green Fluorescent Protein (GFP) family are important and versatile scientific tools used in a range of applications including the labelling of proteins, cells and tissue, high resolution imaging and studying protein-protein interactions. They exhibit a very diverse range of photochemical behaviour. Surprisingly this diversity originates from chromophores which have very similar structures. Accordingly, minor changes to the chromophore structure or to its surroundings in the protein can produce dramatic changes in optical behaviour. In this work the photophysics of the chromophores of the green fluorescent protein, the Kaede fluorescent protein and some related analogues are investigated using ultrafast methods; predominantly the excited state is probed using ultrafast fluorescence up-conversion with a time resolution of better than 50 fs. The excited states of chromophores decay on the ultrafast timescale. The decay will be probed for both isomers of the GFP chromophore, and the mechanism of decay will be modelled, leading to conclusions on the nature of the radiationless coordinate. The derivative of the chromophore generated in the Kaede protein will be studied, and marked differences with the GFP chromophore found. The importance of protein-chromophore interactions in influencing fluorescent protein behaviour will be demonstrated. The effects of chromophore modifications will be revealed through comparison of the analogues and the sensitivity of chromophore photophysics to the hydrogen bonding nature of the solvent will be demonstrated. It will also be shown that the Kaede chromophore undergoes aggregation enhanced fluorescence at relatively low concentrations

The Effect of Conjugation on the Competition Between Internal Conversion and Electron Detachment: A Comparison Between Green Fluorescent and Red Kaede Protein Chromophores

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

Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the β5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling

BLUF Domain Function Does Not Require a Metastable Radical Intermediate 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

Ultrafast studies of the photophysics of cis and trans states of the green fluorescent protein chromophore

Cis–trans photoisomerization is proposed as a key process in the photoswitching of some photoactivatable fluorescent proteins. Here we present ultrafast fluorescence measurements of the model GFP chromophore (HBDI) in the cis state and in a mixture of the cis and trans states. Our results demonstrate that the mean lifetimes of the cis and trans states are remarkably similar. Therefore, the specific isomer of the chromophore cannot be solely responsible for the different photophysics of the bright and dark states of photoactive proteins, which must therefore be due to differential interactions between the different isomers of the chromophore and the protein

Ultrafast ignition of a uni-directional molecular motor

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.