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

Taylor D. Krueger, Longteng Tang, Liangdong Zhu, Isabella L. Breen, Rebekka M. Wachter, Chong Fang

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1644-1652
Number of pages9
JournalAngewandte Chemie - International Edition
Issue number4
StatePublished - Jan 20 2020


  • biophysics
  • conformational trapping
  • dual illumination
  • photoconvertible fluorescent proteins
  • time-resolved spectroscopy

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry


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