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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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This content will become publicly available on November 10, 2026
Dynamics and Mechanism of Off‐ to On‐Switching in Dreiklang a Decoupled Reversibly Switchable Fluorescent Protein
Abstract Dreiklang is a reversibly switchable (rs) fluorescent protein (FP) with a unique off‐state, a UV absorbing hydrated form of the typical FP chromophore. Here we report ultrafast dynamics of the off‐ to on‐state transition in Dreiklang using complementary ultrafast optical and vibrational transient absorption to resolve chromophore driven protein structural dynamics. This approach allows observation of the real‐time response in a protein to bond breaking and forming events. The excited electronic state decays in a nonsingle exponential fashion in tens to hundreds of picoseconds, undergoing photodehydration with a yield of several per‐cent. The primary photoproduct formed is identified as the cis protonated form of the FP chromophore, initially in a perturbed H‐bonded environment. This primary product relaxes on a few microseconds timescale by a mechanism involving changes to a glutamic acid residue and modifications of the amide backbone, possibly involving a carbonyl to imine tautomerization. The temporal and spectral resolution of Dreiklang's photodehydration provides data against which to test quantum chemical calculations of reaction dynamics in proteins and suggests a route to modifying and potentially enhancing its photoswitching properties.
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- Award ID(s):
- 1817837
- PAR ID:
- 10650498
- Publisher / Repository:
- Wiley-VCH
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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