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A detailed study of the rsEGFP2 photodynamics and its variants using time-resolved optical spectroscopies

Abstract : Reversible photo-switchable fluorescent proteins (RSFP) are FP that can be reversibly toggled back and forward between a fluorescent on-state and a non-fluorescent off-state and thus allow to achieve super-resolution in fluorescence microscopy (e.g., in reversible saturable optical fluorescence transition – RESOLFT – microscopy). Even though their photo-physical parameters (switching and fluorescence quantum yield…) are linked to the image resolution and the image acquisition speed, the switching mechanism that controls these parameters is still a matter of debate.This thesis is focused on elucidating the photo-dynamics of rsEGFP2, a negative RSFP variant of the aqua victoria green fluorescent protein (avGFP). The rsEGFP2 is currently the reference fluorescent marker in RESOLFT microscopy. The off to on switching involves a trans-to-cis isomerization and a proton transfer. It was previously highlighted that isomerization dynamics is characterized by a twisted chromophore which is formed at the picosecond time scale and restricted by the close proximity to the Valine 151. The mutation of Valine 151 into alanine (V151A) and leucine (V151L) showed that two different off-conformers exist. Their origin is presumably from a hula-twist and a one-bond-flip on-to-off switching mechanism for V151L and V151A, respectively. In this thesis, we employed electronic and vibrational time-resolved absorption spectroscopy from the femtosecond to the minute time scales to study the photo-dynamics of wild-type rsEGPF2, V151A and V151L. They were combined with the results of time-resolved crystallography obtained by collaborative groups. These two approaches permitted to infer the photo-switching mechanism of rsEGFP2 and its variants. Off-to-on photo-switching quantum yields of 11, 12 and 14% were estimated for WT, V151L and V151A, respectively. Such small differences were rationalized hypothesizing a common trans-to-cis isomerization via a sub-picosecond hula-twist mechanism, followed by microsecond preceding a sub-millisecond-scale multi-step deprotonation. Besides, the thesis also coped with the on-to-off dynamics of 23 other variants of rsEGFP2. From this study, it resulted that fluorescence and switching yield are controlled by the existence of at least two different ground states exhibiting a difference in the fluorescence lifetime of one order of magnitude (150 ps vs 2.3 ns). Overall, the outcomes of these studies will not only contribute to a better understanding of the photo-physics of RSFPs but will also open newer perspectives towards the design of optimized RSFPs for advanced bio-imaging applications.
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Submitted on : Monday, May 16, 2022 - 1:01:20 AM
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Lucas Martinez Uriarte. A detailed study of the rsEGFP2 photodynamics and its variants using time-resolved optical spectroscopies. Analytical chemistry. Université de Lille, 2021. English. ⟨NNT : 2021LILUR006⟩. ⟨tel-03668685⟩

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