Development of the first infrared fluorescent protein with emission above 800 nm – InfraredFP800

Fluorescence cell imaging benefited from the Green Fluorescent Protein revolution in the early 1990’s, which saw the development of genetically-encoded fluorophores covering the whole visible spectrum. 15 years later, another family of fluorescent proteins (FPs) were developed from the chromophore-binding domain of phytochromes, with emission peaks between 670 and 720 nm. Such infrared FPs (IFPs) can be used for whole-body imaging in the optical window of biological tissues (between 660 and 900 nm), in which light is minimally absorbed by haemoglobin and water. Phytochromes are photoreceptors shuffling between a red-light-absorbing (Pr) and a far-red-light-absorbing state (Pfr), whose absorption maxima differ by 50 to 70 nm. As IFPs were developed by stabilizing the chromophore configuration observed in the Pr state, a lead to generate largely red-shifted IFPs would be to stabilize the chromophore configuration observed in the Pfr state. When solving the structure of an IFP from two different crystal forms, we discovered two distinct configurations of the chromophore, one of which resembling that of a Pfr state. We could verify that crystals with this configuration had a red-shifted emission peak. As the change in configuration is due to the repositioning of a short loop that is induced by a crystal contact, we will engineer the protein to stabilize the new chromophore configuration and obtain the first IFP with emission above 800 nm, thus extending the possibilities of multicolour imaging in the optical window. To this end, we will use high-pressure crystallography, structure prediction using artificial intelligence and molecular dynamics simulations in order to further our understanding of protein dynamics.