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Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein
Author(s) -
Krueger Taylor D.,
Tang Longteng,
Zhu Liangdong,
Breen Isabella L.,
Wachter Rebekka M.,
Fang Chong
Publication year - 2020
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201911379
Subject(s) - chromophore , green fluorescent protein , fluorescence , photochemistry , fluorescent protein , rational design , raman spectroscopy , chemistry , protein dynamics , materials science , molecular dynamics , nanotechnology , optics , physics , computational chemistry , biochemistry , gene
The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light‐induced processes. We implemented time‐resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP‐like protein LEA, involving semi‐trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual‐illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge‐H bending motions enables rational design of functional proteins. With the improved H‐bonding network and structural motions, the photoexcited chromophore could increase the photoswitching‐aided photoconversion while reducing trapped species.