Defense of thesis Cédric Mittelheisser

Defense of thesis Cédric Mittelheisser - laboratory LASIRe

Abstract :

mong all the applications of fluorescent proteins, their use as markers in fluorescence microscopy is undoubtedly the most widespread, a discovery recognized by the Nobel Prize in Chemistry in 2008. In recent years, reversible switchable fluorescent proteins (RSFPs) have emerged, characterized by a reversible, photo-induced switching between a fluorescent (ON) state and a non-fluorescent (OFF) state. Their relevance lies in their use in super-resolution fluorescence microscopy (nanoscopy) to achieve nanometric spatial resolutions, a breakthrough that earned the Nobel Prize in Chemistry in 2014. The imaging acquisition parameters, speed, and resolution are closely tied to the photophysical properties and switching photodynamics of these proteins. Today, efforts are focused on developing photo-switchable fluorescent proteins that operate in the near-infrared (NIR) range, which offers ideal wavelengths for deep-tissue biological imaging. Professor Stefan Jakobs' group at the Max Planck Institute for Multidisciplinary Sciences in Göttingen has developed PENELOPE, the first RSFP to function in NIR nanoscopy (λex = 690 nm, λem = 720 nm) with an in vitro millisecond thermal recovery. PENELOPE is a derivative of the wild-type bacteriophytochrome from Deinococcus radiodurans (Dr-PSM). The stable form of Dr-PSM absorbs in the red (Pr, ON state) and, upon irradiation, converts into a form that absorbs in the near-infrared (Pfr, OFF state). Its thermal recovery takes several days. Developing new mutants based on PENELOPE requires a deep understanding of its photo-switching mechanism, particularly to identify the species that control its fluorescence and switching quantum yields, as well as its rapid thermal recovery. The photo-switching from Pr to Pfr in wild-type bacteriophytochromes involves multiple processes, including a picosecond cis-trans isomerization of the biliverdin chromophore, millisecond deprotonation and reprotonation steps, and structural changes in the protein, leading to the formation of the final Pfr state within hundreds of milliseconds. The photodynamics thus involves several excited and intermediate states, with lifetimes spanning fifteen orders of magnitude, from femtoseconds to seconds. In this thesis, the photodynamics of PENELOPE was studied using time-resolved optical spectroscopy, including fluorescence and transient absorption UV-Vis-NIR, covering a time range from femtoseconds to seconds. A comparison was made with variants of the wild-type protein exhibiting (i) accelerated thermal recovery (Dr-CBDmono) or (ii) fluorescence without photo-switching capabilities (SNIFP). This comparison allowed for the identification of two specific excited states that control fluorescence and isomerization, respectively. The photoactivation of PENELOPE from the ON state (Pr state) is characterized by the formation of the OFF state within a few milliseconds, with an absorption and Raman signature similar to that of the Pfr precursor in the wild-type protein. This OFF state exhibits a thermal recovery to Pr over a period of several hours for the purified protein, which is accelerated by several orders of magnitude in vitro, i.e., when expressed in E. coli. The results of these studies will contribute to the design of new bacteriophytochrome variants for deep-tissue nanoscopy.

Keywords : Photo-switchable,Near Infra-Red,Phytochrome,Fluorescent Protein,Photo-Dynamics,Spectroscopy