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To twist or not to twist: From chromophore structure to dynamics inside engineered photoconvertible and photoswitchable fluorescent proteins

https://doi.org/10.1002/pro.4517

Krueger, Taylor D. ; Tang, Longteng ; Chen, Cheng ; Zhu, Liangdong ; Breen, Isabella L. ; Wachter, Rebekka M. ; Fang, Chong ( January 2023 , Protein Science)

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Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

https://doi.org/10.1002/ange.201911379

Krueger, Taylor D. ; Tang, Longteng ; Zhu, Liangdong ; Breen, Isabella L. ; Wachter, Rebekka M. ; Fang, Chong ( November 2019 , Angewandte Chemie)

Abstract
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.

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Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

https://doi.org/10.1002/anie.201911379

Krueger, Taylor D. ; Tang, Longteng ; Zhu, Liangdong ; Breen, Isabella L. ; Wachter, Rebekka M. ; Fang, Chong ( November 2019 , Angewandte Chemie International Edition)

Abstract
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.

more » « less