Photoswitching mechanisms and environmental dependencies of fluorescent protein markers – Photoswitch-NMR

Photo-switchable fluorescent protein markers provide crucial tools for a wide range of applications in advanced fluorescence microscopy such as super-resolution and multiplexed microscopies. A major challenge for the optimal use of these molecules in cellular applications is the dependency of their photophysical properties such as photoswitching kinetics and contrast, fluorescence brightness, and photo stability on environmental conditions. We have recently set up an in-situ illumination device that allows NMR investigation of photo-switchable proteins in their different photo-induced states. In this project we will exploit the atomic-resolution information obtained from solution NMR spectroscopy, combined with crystallographic structures, optical measurements (fluorescence and absorbance), and molecular dynamics simulations, to derive mechanistic insight how environmental changes influence the photophysical properties of 2 classes of prominent fluorescent markers: (i) Reversibly switchable and photo-convertible (Green to Red) fluorescent proteins (RSFPs) derived from Green Fluroescent Protein (GFP) that possess an endogenous chromophore, and (ii) Fluorescence activating and Absortpion Shifting Tag (FAST) that are hybrid systems composed of an engineered small protein and a synthetic organic fluorophore that dynamically and reversibly binds to the protein tag. Our study aims at providing a better understanding of the underlying mechanisms of the photoswitching properties observed in cellular environments, that will open up new ways on how to improve the photophysical properties of these markers for a variety of applications.