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. 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.
Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium. virgile.adam@ibs.fr
Virgile
V
Adam
Benjamien
B
Moeyaert
Charlotte C
CC
David
Hideaki
H
Mizuno
Mickaël
M
Lelimousin
Peter
P
Dedecker
Ryoko
R
Ando
Atsushi
A
Miyawaki
Jan
J
Michiels
Yves
Y
Engelborghs
Johan
J
Hofkens
D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation; D010777:Photochemistry / Q000379:methods; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins