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1. Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor

   https://doi.org/10.3390/ijms22010445  

   Tang, Longteng; Zhang, Shuce; Zhao, Yufeng; Rozanov, Nikita D.; Zhu, Liangdong; Wu, Jiahui; Campbell, Robert E.; Fang, Chong (January 2021, International Journal of Molecular Sciences)

   null (Ed.)

   Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths. 

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2. Deciphering ultrafast structural dynamics of the red Kaede chromophore in solution and a photoconvertible fluorescent protein

   https://doi.org/10.1063/5.0268655  

   Krueger, Taylor D; Chen, Cheng; Fang, Chong (April 2025, The Journal of Chemical Physics)

   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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3. Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy

   https://doi.org/10.3390/ijms241511991  

   Chen, Cheng; Henderson, J. Nathan; Ruchkin, Dmitry A.; Kirsh, Jacob M.; Baranov, Mikhail S.; Bogdanov, Alexey M.; Mills, Jeremy H.; Boxer, Steven G.; Fang, Chong (August 2023, International Journal of Molecular Sciences)

   The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore–environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore–environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions. 

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4. Photogrammetry of Ultrafast Excited-State Intramolecular Proton Transfer Pathways in the Fungal Pigment Draconin Red

   https://doi.org/10.3390/molecules28083506  

   Solaris, Janak; Krueger, Taylor D.; Chen, Cheng; Fang, Chong (April 2023, Molecules)

   Proton transfer processes of organic molecules are key to charge transport and photoprotection in biological systems. Among them, excited-state intramolecular proton transfer (ESIPT) reactions are characterized by quick and efficient charge transfer within a molecule, resulting in ultrafast proton motions. The ESIPT-facilitated interconversion between two tautomers (PS and PA) comprising the tree fungal pigment Draconin Red in solution was investigated using a combination of targeted femtosecond transient absorption (fs-TA) and excited-state femtosecond stimulated Raman spectroscopy (ES-FSRS) measurements. Transient intensity (population and polarizability) and frequency (structural and cooling) dynamics of –COH rocking and –C=C, –C=O stretching modes following directed stimulation of each tautomer elucidate the excitation-dependent relaxation pathways, particularly the bidirectional ESIPT progression out of the Franck–Condon region to the lower-lying excited state, of the intrinsically heterogeneous chromophore in dichloromethane solvent. A characteristic overall excited-state PS-to-PA transition on the picosecond timescale leads to a unique “W”-shaped excited-state Raman intensity pattern due to dynamic resonance enhancement with the Raman pump–probe pulse pair. The ability to utilize quantum mechanics calculations in conjunction with steady-state electronic absorption and emission spectra to induce disparate excited-state populations in an inhomogeneous mixture of similar tautomers has broad implications for the modeling of potential energy surfaces and delineation of reaction mechanisms in naturally occurring chromophores. Such fundamental insights afforded by in-depth analysis of ultrafast spectroscopic datasets are also beneficial for future development of sustainable materials and optoelectronics. 

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5. Ultrafast spectroscopic signatures for off -to- on photoswitchable species in a green-to-red photoconvertible fluorescent protein

   https://doi.org/10.1039/D5SC06279A  

   Krueger, Taylor D; Chen, Cheng; Fang, Chong (January 2026, Chemical Science)

   Ultrafast electronic and Raman spectroscopies on least evolved ancestor (LEA) fluorescent proteins reveal a distorted photoswitchedoffstate returning toonstate upon photoexcitationviaproton transfer and isomerization, hindering photoconversion. Photoconvertible fluorescent proteins (pcFPs) have greatly advanced life sciences and cellular imaging with sub-diffraction resolution. A subset of Kaede-like pcFPs can reversibly photoswitch and irreversibly photoconvert, which yield intriguing properties for sophisticated bioimaging, yet blinking may complicate image analysis. Many investigations on such pcFPs lack transient information that can dictate their optical properties, especially on ultrafast timescales. We study a family of ancestrally derived pcFPs with varying photoconversion and photoswitching efficiencies based on the least evolved ancestor (LEA). With ultrafast electronic and vibrational spectroscopies that complement steady-state measurements, we dissect the primary events upon near-UV excitation of the native and photoswitched neutral chromophores, which initiates both off → on photoswitching and green-to-red photoconversion. We demonstrate that cis → trans isomerization underlying negative photoswitching occurs the fastest in acidic buffers upon green light irradiation, which forms a distorted neutral off state proportional to the initial green cis anionic population. Femtosecond transient absorption measurements reveal that this dynamic off state rapidly photoswitches back to the bright on state upon near-UV excitation, in contrast to the native form. With various mutants and photoinduced states, we find that off → on photoswitching likely involves excited state proton transfer from the distorted chromophore, which competes with photoconversion. In contrast, femtosecond stimulated Raman spectroscopy (FSRS) of the much less photoswitchable LEA-A69T tracks the efficient nonradiative relaxation of the native cis neutral chromophore. We propose rational design strategies to inhibit off → on photoswitching while improving the photoconversion efficiency of both neutral states. This work is envisioned to inspire more dynamic investigations of diverse photochromic FPs on ultrafast timescales. 

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