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

• 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
• ISSN: 1074-5521; 1879-1301
• DOI: 10.1016/j.chembiol.2011.08.007

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