Structural insight into photobleaching mechanisms of reversible photoswitchable fluorescent proteins

Abstract : The discovery of phototransformable FPs (PTFPs) from Anthozoa species, thanks totheir photophysical properties, has opened a large field in biological fluorescence imaging.One of the PTFPs’ sub-groups consists of Reversible Photoswitchable Fluorescent Proteins(RSFPs), which can be reversibly switched between nonfluorescent and fluorescent states.Photobleaching is the permanent loss of the fluorescence-emitting capacity under excitation,which is a common phenomenon among all the fluorescent molecules. Photobleaching has alarge impact on the microscopy image quality, notably on super-resolution imaging.Photoswitchable fluorescent proteins have a tendency to lose performance within everyswitching cycle, a process referred to as “photofatigue”. Our interest of study is focused onthe photobleaching mechanisms of RSFPs.We have reported the crystallographic structure of photobleached IrisFP under highand low illumination intensity at room temperature as well as its spectroscopic modifications.We found that different illumination intensities can result in different photobleachingpathways. Under low illumination intensity, an oxygen-dependent photobleaching pathwaywas evidenced. Structural modifications induced by singlet-oxygen production within thechromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 andCys171, locking the chromophore in a nonfluorescent protonated state. Under highillumination intensity, a completely different, oxygen-independent photobleaching pathwaywas found. The conserved Glu212 underwent decarboxylation concomitantly with anextensive rearrangement of the H-bond network around the chromophore, and an sp2-to-sp3hybridization change of the carbon atom bridging the chromophore cyclic moieties wasobserved. As Met159 is the key residue involved in low-intensity illumination photobleaching,we have then mutated Met159 into Alanine in order to avoid sulfoxidation. We found that theIrisFP-M159A mutant display an enhanced photostability in solution, in PVA gel and inE.coli cells.
Complete list of metadatas

https://tel.archives-ouvertes.fr/tel-01097264
Contributor : Abes Star <>
Submitted on : Thursday, May 12, 2016 - 5:23:02 PM
Last modification on : Friday, April 5, 2019 - 1:01:56 AM

File

DUAN_2014_diffusion.pdf
Version validated by the jury (STAR)

Identifiers

  • HAL Id : tel-01097264, version 2

Collections

Citation

Chenxi Duan. Structural insight into photobleaching mechanisms of reversible photoswitchable fluorescent proteins. Biomolecules [q-bio.BM]. Université de Grenoble, 2014. English. ⟨NNT : 2014GRENV034⟩. ⟨tel-01097264v2⟩

Share

Metrics

Record views

603

Files downloads

611