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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Mapping Structural Dynamics of Proteins with Femtosecond Stimulated Raman Spectroscopy
The structure–function relationships of biomolecules have captured the interest and imagination of the scientific community and general public since the field of structural biology emerged to enable the molecular understanding of life processes. Proteins that play numerous functional roles in cellular processes have remained in the forefront of research, inspiring new characterization techniques. In this review, we present key theoretical concepts and recent experimental strategies using femtosecond stimulated Raman spectroscopy (FSRS) to map the structural dynamics of proteins, highlighting the flexible chromophores on ultrafast timescales. In particular, wavelength-tunable FSRS exploits dynamic resonance conditions to track transient-species-dependent vibrational motions, enabling rational design to alter functions. Various ways of capturing excited-state chromophore structural snapshots in the time and/or frequency domains are discussed. Continuous development of experimental methodologies, synergistic correlation with theoretical modeling, and the expansion to other nonequilibrium, photoswitchable, and controllable protein systems will greatly advance the chemical, physical, and biological sciences.
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- Award ID(s):
- 1455353
- PAR ID:
- 10159263
- Date Published:
- Journal Name:
- Annual Review of Physical Chemistry
- Volume:
- 71
- Issue:
- 1
- ISSN:
- 0066-426X
- Page Range / eLocation ID:
- 239 to 265
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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