PhD thesis : Mechanistic studies of photoactivatable fluorescent proteins: a combined approach by crystallography and spectroscopy
Since the discovery of the green fluorescent protein (GFP) in 1962, many developments allowed improving the use of this naturally light-emitting protein as a powerful tool for tracking proteins or organelles of interest within living cells and organisms. At the beginning of the 21st century, the discovery of photoactivatable fluorescent proteins (PAFPs), notably from Anthozoan species, triggered a revolution in the field of FP technology. Some PAFPs are capable of being irreversibly photoconverted from a green- to a red-emitting form while other ones can be reversibly switched on and off, depending on specific excitation wavelengths. These proteins are being extensively used in optical microscopy techniques, particularly in “nanoscopy”, which provides optical resolution 10 fold beyond the theoretical Abbe limit. In order to further develop these techniques, notably in term of time-resolution, the need to obtain brighter fluorescent probes that photoconvert or photoswitch efficiently is crucial. At the same time, fluorescent highlighters generally need to be monomeric and photostable. In order to better understand the mechanisms of phototransformations in PAFPs, three members of the family have been studied: EosFP, Dendra2 and IrisFP. The phenomena of green-to-red photoconversion, reversible photoswitching and non-reversible photobleaching have been studied by a combination of X-ray crystallography and microspectrophotometry using the Cryobench laboratory of the ESRF/IBS. Together, the results have allowed us to propose a mechanism for the photoconversion of EosFP and Dendra2 and to discover and characterize IrisFP, the first PAFP combining both properties of photoconversion and photoswitching. The structural modifications of the chromophore associated with an X-ray induced radical state, likely to be involved in the photobleaching pathway of PAFPs, were also characterized.
keywords: Fluorescent proteins – photoconversion – photoswitching – photobleaching – protein crystallography – microspectrophotometry – synchrotron – Cryobench