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Photoconvertible fluorescent proteins (pcFPs) have enabled exquisite images of cellular structures due to their genetic encodability and red-shifted emission with high brightness, hence receiving increased traction in the field. However, the red form of Kaede-like pcFPs after photoconversion remains underexplored. We implemented ultrafast electronic and vibrational spectroscopies on the red Kaede chromophore in solution vs the protein pocket of the least-evolved ancestor (LEA, a Kaede-like green-to-red pcFP) to gain crucial insights into the photophysical processes of the chromophore. The measured fluorescence quantum yield (FQY) values were correlated with ultrafast dynamics to reveal that hydrogen-bonding interactions with the solvent can quench the excited-state Kaede in solution. A viscosity-dependent sub-ps decay indicates nonradiative relaxation involving swift chromophore conformational motions. Femtosecond transient absorption and stimulated Raman spectroscopy (FSRS) reveal an additional ∼1 ps decay of the photoconverted red form of LEA that is absent in green LEA before photoconversion. Transient structural dynamics from FSRS elucidate this decay to involve the phenolate and imidazolinone ring twists that are implicated during cis → trans isomerization and on → off photoswitching in phototransformable fluorescent proteins (FPs). Compared to green-emitting species, the FQY of red LEA (∼0.58) and many other red FPs are often reduced, limiting their applications in modern bioimaging techniques. By shining more light on the often overlooked photoconverted form of pcFPs with ultrafast spectroscopies, we envision such essential mechanistic insights to enable a bottom-up approach for rationally improving the brightness of red-emitting LEA and many other controllable bioprobes, including FPs.
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Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein
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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- Award ID(s):
- 1817949
- PAR ID:
- 10125798
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 59
- Issue:
- 4
- ISSN:
- 1433-7851
- Page Range / eLocation ID:
- p. 1644-1652
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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