Structural characterisation of novel GFP-like and phytochrome-derived fluorescent proteins

Overview & Synopsis

Since the cloning of the Green Fluorescent Protein (GFP) in 1992 fluorescent proteins (FPs) have increasingly become essential tools in cell imaging. GFP was first discovered in the jellyfish Aequorea victoria, then homologues were found in other types of organisms such as corals, sea anemones, lancelets and small crustaceans, forming the family of GFP-like FPs. A unique feature of GFP-like proteins is the autocatalytic formation of the chromophore, the light-absorbing and light-emitting part of the protein, from three consecutive amino acids located at the centre of the β-barrel structure of the protein. The family of GFP-like FPs have fluorescence emission maxima that cover the entire visible light spectrum from deep blue to far red. In the late 2000s, another type of FPs has been derived from phytochromes, a family of red-light photoreceptors that use bilins as chromophore. The interest of these is that their fluorescence excitation and emission spectra is in the near-infrared region of the light spectrum. This region is part of the so-called ‘optical window’ of living tissues, in which light absorption and scattering by haemoglobin, water and lipids is minimized and thus should be preferred for whole-body fluorescence imaging . These FPs have been called NIR FPs. In this PhD work, I have solved the crystallographic structures of three GFP-like FPs (one very bright green FP, one chromoprotein and one weakly red fluorescent FP) and three NIR FPs derived from a monomeric phytochrome. The structural information gained on the nature and environment of the chromophores has allowed me to propose explanations for their peculiar spectroscopic properties. In particular the structure of the chromoprotein revealed the existence for the first time of a chromophore which forms a covalent bond with a nearby cysteine residue, leading to a large red-shift of the UV-vis absorption maximum.