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1. Ultrafast internal conversion dynamics of bilirubin bound to UnaG and its N57A mutant

   https://doi.org/10.1039/c8cp07553k  

   Cao, Xiaodan ; Zhang, Changcheng ; Gao, Ziheng ; Liu, Yangyi ; Zhao, Yuzheng ; Yang, Yi ; Chen, Jinquan ; Jimenez, Ralph ; Xu, Jianhua ( January 2019 , Physical Chemistry Chemical Physics)

   Fluorescent proteins (FPs) have become fundamental tools for live cell imaging. Most FPs currently used are members of the green fluorescent protein super-family, but new fluorophores such as bilin-FPs are being developed and optimized. In particular, the UnaG FP incorporates bilirubin (BR) as a chromophore, enhancing its fluorescence quantum yield by three orders of magnitude relative to that in solution. To investigate the mechanism of this dramatic enhancement and provide a basis for further engineering of UnaG and other tetrapyrrole-based fluorophores, we performed picosecond fluorescence and femtosecond transient absorption measurements of BR bound to UnaG and its N57A site-directed mutant. The dynamics of wt-UnaG, which has a fluorescence QY of 0.51, are largely homogeneous, showing an excited state relaxation of ∼200 ps, and a 2.2 ns excited-state lifetime decay with a kinetic isotope effect (KIE) of 1.1 for D 2 O vs. H 2 O buffer. In contrast, for UnaG N57A (fluorescence QY 0.01) the results show a large spectral inhomogeneity with excited state decay timescales of 47 and 200 ps and a KIE of 1.4. The non-radiative deactivation of the excited state is limited by proton transfer. The loss of direct hydrogen bonds to the endo -vinyl dipyrrinone moietymore »of BR leads to high flexibility and structural heterogeneity of UnaG N57A, as seen in the X-ray crystal structure.« less
2. Shedding light on ultrafast ring-twisting pathways of halogenated GFP chromophores from the excited to ground state

   https://doi.org/10.1039/D1CP02140K  

   Boulanger, Sean A. ; Chen, Cheng ; Tang, Longteng ; Zhu, Liangdong ; Baleeva, Nadezhda S. ; Myasnyanko, Ivan N. ; Baranov, Mikhail S. ; Fang, Chong ( July 2021 , Physical Chemistry Chemical Physics)

   Since green fluorescent protein (GFP) has revolutionized molecular and cellular biology for about three decades, there has been a keen interest in understanding, designing, and controlling the fluorescence properties of GFP chromophore ( i.e. , HBDI) derivatives from the protein matrix to solution. Amongst these cross-disciplinary efforts, the elucidation of excited-state dynamics of HBDI derivatives holds the key to correlating the light-induced processes and fluorescence quantum yield (FQY). Herein, we implement steady-state electronic spectroscopy, femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and quantum calculations to study a series of mono- and dihalogenated HBDI derivatives (X = F, Cl, Br, 2F, 2Cl, and 2Br) in basic aqueous solution, gaining new insights into the photophysical reaction coordinates. In the excited state, the halogenated “floppy” chromophores exhibit an anti-heavy atom effect, reflected by strong correlations between FQY vs. Franck–Condon energy ( E FC ) or Stokes shift, and k nr vs. E FC , as well as a swift bifurcation into the I-ring (major) and P-ring (minor) twisting motions. In the ground state, both ring-twisting motions become more susceptible to sterics and exhibit spectral signatures from the halogen-dependent hot ground-state absorption band decay in TA data. We envision this type ofmore »systematic analysis of the halogenated HBDI derivatives to provide guiding principles for the site-specific modification of GFP chromophores, and expand design space for brighter and potentially photoswitchable organic chemical probes in aqueous solution with discernible spectral signatures throughout the photocycle.« less
3. Ultrafast Spectroscopies of Nitrophenols and Nitrophenolates in Solution: From Electronic Dynamics and Vibrational Structures to Photochemical and Environmental Implications

   https://doi.org/10.3390/molecules28020601  

   Bailey-Darland, Sullivan ; Krueger, Taylor D. ; Fang, Chong ( January 2023 , Molecules)

   Nitrophenols are a group of small organic molecules with significant environmental implications from the atmosphere to waterways. In this work, we investigate a series of nitrophenols and nitrophenolates, with the contrasting ortho-, meta-, and para-substituted nitro group to the phenolic hydroxy or phenolate oxygen site (2/3/4NP or NP−), implementing a suite of steady-state and time-resolved spectroscopic techniques that include UV/Visible spectroscopy, femtosecond transient absorption (fs-TA) spectroscopy with probe-dependent and global analysis, and femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations. The excitation-dependent (400 and 267 nm) electronic dynamics in water and methanol, for six protonated or deprotonated nitrophenol molecules (three regioisomers in each set), enable a systematic investigation of the excited-state dynamics of these functional “nanomachines” that can undergo nitro-group twisting (as a rotor), excited-state intramolecular or intermolecular proton transfer (donor–acceptor, ESIPT, or ESPT), solvation, and cooling (chromophore) events on molecular timescales. In particular, the meta-substituted compound 3NP or 3NP− exhibits the strongest charge-transfer character with FSRS signatures (e.g., C–N peak frequency), and thus, does not favor nitroaromatic twist in the excited state, while the ortho-substituted compound 2NP can undergo ESIPT in water and likely generate nitrous acid (HONO) after 267 nm excitation. The delineated mechanistic insights into themore »nitro-substituent-location-, protonation-, solvent-, and excitation-wavelength-dependent effects on nitrophenols, in conjunction with the ultraviolet-light-induced degradation of 2NP in water, substantiates an appealing discovery loop to characterize and engineer functional molecules for environmental applications.« less
4. Ultrafast spectroscopy of biliverdin dimethyl ester in solution: pathways of excited-state depopulation

   https://doi.org/10.1039/D0CP02971H  

   Liu, Yangyi ; Chen, Zhuang ; Wang, Xueli ; Cao, Simin ; Xu, Jianhua ; Jimenez, Ralph ; Chen, Jinquan ( September 2020 , Physical Chemistry Chemical Physics)

   Biliverdin is a bile pigment that has a very low fluorescence quantum yield in solution, but serves as a chromophore in far-red fluorescent proteins being developed for bio-imaging. In this work, excited-state dynamics of biliverdin dimethyl ether (BVE) in solvents were investigated using femtosecond (fs) and picosecond (ps) time-resolved absorption and fluorescence spectroscopy. This study is the first fs timescale investigation of BVE in solvents, and therefore revealed numerous dynamics that were not resolved in previous, 200 ps time resolution measurements. Viscosity- and isotope-dependent experiments were performed to identify the contributions of isomerization and proton transfer to the excited-state dynamics. In aprotic solvents, a ∼2 ps non-radiative decay accounts for 95% of the excited-state population loss. In addition, a minor ∼30 ps emissive decay pathway is likely associated with an incomplete isomerization process around the C15C16 double bond that results in a flip of the D-ring. In protic solvents, the dynamics are more complex due to hydrogen bond interactions between solute and solvent. In this case, the ∼2 ps decay pathway is a minor channel (15%), whereas ∼70% of the excited-state population decays through an 800 fs emissive pathway. The ∼30 ps timescale associated with isomerization is also observed inmore »protic solvents. The most significant difference in protic solvents is the presence of a >300 ps timescale in which BVE can decay through an emissive state, in parallel with excited-state proton transfer to the solvent. Interestingly, a small fraction of a luminous species, which we designate lumin-BVE (LBVE), is present in protic solvents.« less
5. Excitation ratiometric chloride sensing in a standalone yellow fluorescent protein is powered by the interplay between proton transfer and conformational reorganization

   https://doi.org/10.1039/D1SC00847A  

   Chen, Cheng ; Tutol, Jasmine N. ; Tang, Longteng ; Zhu, Liangdong ; Ong, Whitney S. ; Dodani, Sheel C. ; Fang, Chong ( January 2021 , Chemical Science)

   Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl − ). To date, such sensors have been limited to the Aequorea victoria green fluorescent protein (avGFP) family, and fusions with other FPs have unlocked ratiometric imaging applications. Recently, we identified the yellow fluorescent protein from jellyfish Phialidium sp. (phiYFP) as a fluorescent turn-on, self-ratiometric Cl − sensor. To elucidate its working mechanism as a rare example of a single FP with this capability, we tracked the excited-state dynamics of phiYFP using femtosecond transient absorption (fs-TA) spectroscopy and target analysis. The photoexcited neutral chromophore undergoes bifurcated pathways with the twisting-motion-induced nonradiative decay and barrierless excited-state proton transfer. The latter pathway yields a weakly fluorescent anionic intermediate , followed by the formation of a red-shifted fluorescent state that enables the ratiometric response on the tens of picoseconds timescale. The redshift results from the optimized π–π stacking between chromophore Y66 and nearby Y203, an ultrafast molecular event. The anion binding leads to an increase of the chromophore p K a and ESPT population, and the hindrance of conversion. The interplay between these two effects determines the turn-on fluorescence response to halides such as Cl −more »but turn-off response to other anions such as nitrate as governed by different binding affinities. These deep mechanistic insights lay the foundation for guiding the targeted engineering of phiYFP and its derivatives for ratiometric imaging of cellular chloride with high selectivity.« less