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Rational design of enhanced photoresistance in a photoswitchable fluorescent protein.
Methods Appl Fluoresc 2015; 3(1):014004MA

Abstract

Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions.

Authors+Show Affiliations

Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France. CNRS, IBS, F-38044 Grenoble, France. CEA, IBS, F-38044 Grenoble, France.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

29148481

Citation

Duan, Chenxi, et al. "Rational Design of Enhanced Photoresistance in a Photoswitchable Fluorescent Protein." Methods and Applications in Fluorescence, vol. 3, no. 1, 2015, p. 014004.
Duan C, Byrdin M, El Khatib M, et al. Rational design of enhanced photoresistance in a photoswitchable fluorescent protein. Methods Appl Fluoresc. 2015;3(1):014004.
Duan, C., Byrdin, M., El Khatib, M., Henry, X., Adam, V., & Bourgeois, D. (2015). Rational design of enhanced photoresistance in a photoswitchable fluorescent protein. Methods and Applications in Fluorescence, 3(1), p. 014004. doi:10.1088/2050-6120/3/1/014004.
Duan C, et al. Rational Design of Enhanced Photoresistance in a Photoswitchable Fluorescent Protein. Methods Appl Fluoresc. 2015 Jan 16;3(1):014004. PubMed PMID: 29148481.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rational design of enhanced photoresistance in a photoswitchable fluorescent protein. AU - Duan,Chenxi, AU - Byrdin,Martin, AU - El Khatib,Mariam, AU - Henry,Xavier, AU - Adam,Virgile, AU - Bourgeois,Dominique, Y1 - 2015/01/16/ PY - 2017/11/18/entrez PY - 2015/1/16/pubmed PY - 2015/1/16/medline SP - 014004 EP - 014004 JF - Methods and applications in fluorescence JO - Methods Appl Fluoresc VL - 3 IS - 1 N2 - Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions. SN - 2050-6120 UR - https://www.unboundmedicine.com/medline/citation/29148481/Rational_design_of_enhanced_photoresistance_in_a_photoswitchable_fluorescent_protein_ L2 - https://doi.org/10.1088/2050-6120/3/1/014004 DB - PRIME DP - Unbound Medicine ER -