Rational Design of Photoconvertible and Biphotochromic Fluorescent Proteins for Advanced Microscopy Applications

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. Iri...

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Published in:	Chemistry & biology Vol. 18; no. 10; pp. 1241 - 1251
Main Authors:	Adam, Virgile, Moeyaert, Benjamien, David, Charlotte C., Mizuno, Hideaki, Lelimousin, Mickaël, Dedecker, Peter, Ando, Ryoko, Miyawaki, Atsushi, Michiels, Jan, Engelborghs, Yves, Hofkens, Johan
Format:	Journal Article
Language:	English
Published:	United States Elsevier Ltd 28-10-2011 Elsevier
Subjects:	Biochemistry, Molecular Biology Biophysics Fluorescent Dyes - chemistry Green Fluorescent Proteins - chemistry Green Fluorescent Proteins - genetics HeLa Cells Humans Life Sciences Luminescent Proteins - chemistry Luminescent Proteins - genetics Microscopy, Fluorescence - methods Models, Molecular Mutation Photochemistry - methods Protein Conformation Protein Engineering - methods Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Spectrometry, Fluorescence Structure-Activity Relationship
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Abstract	Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications. ► Fluorescent proteins capable of four-way highlighting are rationally designed ► Interesting mutants are characterized through spectroscopy and modeling ► New fluorescent proteins are successfully applied in advanced microscopy
AbstractList	Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications. Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications. ► Fluorescent proteins capable of four-way highlighting are rationally designed ► Interesting mutants are characterized through spectroscopy and modeling ► New fluorescent proteins are successfully applied in advanced microscopy
Author	Michiels, Jan Lelimousin, Mickaël Moeyaert, Benjamien Hofkens, Johan Ando, Ryoko Engelborghs, Yves Mizuno, Hideaki Dedecker, Peter Miyawaki, Atsushi Adam, Virgile David, Charlotte C.
Author_xml	– sequence: 1 givenname: Virgile surname: Adam fullname: Adam, Virgile email: virgile.adam@ibs.fr organization: Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium – sequence: 2 givenname: Benjamien surname: Moeyaert fullname: Moeyaert, Benjamien organization: Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium – sequence: 3 givenname: Charlotte C. surname: David fullname: David, Charlotte C. organization: Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium – sequence: 4 givenname: Hideaki surname: Mizuno fullname: Mizuno, Hideaki organization: Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium – sequence: 5 givenname: Mickaël surname: Lelimousin fullname: Lelimousin, Mickaël organization: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK – sequence: 6 givenname: Peter surname: Dedecker fullname: Dedecker, Peter organization: Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium – sequence: 7 givenname: Ryoko surname: Ando fullname: Ando, Ryoko organization: Laboratory for Cell Function Dynamics, RIKEN Brain Science Institute, Wako-city, Saitama, 351-0198, Japan – sequence: 8 givenname: Atsushi surname: Miyawaki fullname: Miyawaki, Atsushi organization: Laboratory for Cell Function Dynamics, RIKEN Brain Science Institute, Wako-city, Saitama, 351-0198, Japan – sequence: 9 givenname: Jan surname: Michiels fullname: Michiels, Jan organization: Centre of Microbial and Plant Genetics, Department of Microbial and Molecular Systems, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium – sequence: 10 givenname: Yves surname: Engelborghs fullname: Engelborghs, Yves organization: Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium – sequence: 11 givenname: Johan surname: Hofkens fullname: Hofkens, Johan organization: Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium
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Snippet	Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these...
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SubjectTerms	Biochemistry, Molecular Biology Biophysics Fluorescent Dyes - chemistry Green Fluorescent Proteins - chemistry Green Fluorescent Proteins - genetics HeLa Cells Humans Life Sciences Luminescent Proteins - chemistry Luminescent Proteins - genetics Microscopy, Fluorescence - methods Models, Molecular Mutation Photochemistry - methods Protein Conformation Protein Engineering - methods Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Spectrometry, Fluorescence Structure-Activity Relationship
Title	Rational Design of Photoconvertible and Biphotochromic Fluorescent Proteins for Advanced Microscopy Applications
URI	https://dx.doi.org/10.1016/j.chembiol.2011.08.007 https://www.ncbi.nlm.nih.gov/pubmed/22035793 https://search.proquest.com/docview/901304220 https://hal.science/hal-02389699
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