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1. 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)

   null (Ed.)

   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 − 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. 

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2. Discovery and Characterization of a Bilirubin Inducible Green Fluorescent Protein From the Moray Eel Gymnothorax zonipectis

   https://doi.org/10.3389/fmars.2021.678571  

   Guarnaccia, Andrew M. ; Krivoshik, Sara Rose ; Sparks, John S. ; Gruber, David F. ; Gaffney, Jean P. ( September 2021 , Frontiers in Marine Science)

   Since the initial discovery of Aqueoria victoria ’s green fluorescence off the coast of Washington’s Puget Sound, biofluorescent marine organisms have been found across the globe. The variety of colors of biofluorescence as well as the variability in the organisms that exhibit this fluorescence is astounding. The mechanisms of biofluorescence in marine organisms are also variable. To fluoresce, some organisms use fluorescent proteins, while others use small molecules. In eels, green biofluorescence was first identified in Anguilla japonica . The green fluorescence in A. japonica was discovered to be caused by a fatty acid binding protein (UnaG) whose fluorescence is induced by the addition of bilirubin. Members of this class of proteins were later discovered in Kaupichthys eels (Chlopsid FP I and Chlopsid FP II). Here, we report the discovery and characterization of the first member of this class of green fluorescent fatty acid binding proteins from the moray eel Gymnothorax zonipectis . This protein, GymFP, is 15.6 kDa with a fluorescence excitation at 496 nm and an emission maximum at 532 nm upon addition of bilirubin. GymFP is 61% homologous to UnaG and 47% homologous to Chlopsid FP I. Here, we report de novo transcriptome assembly, protein expression, and fluorescence spectroscopic characterization of GymFP. These findings extend the fluorescent fatty acid binding proteins into a third family of true eels (Anguilliformes). 

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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. New Aequorea Fluorescent Proteins for Cell-Free Bioengineering

   https://doi.org/10.1021/acssynbio.3c00057  

   Deich, Christopher ; Gaut, Nathaniel J. ; Sato, Wakana ; Engelhart, Aaron E. ; Adamala, Katarzyna P. ( April 2023 , ACS Synthetic Biology)

   Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing, and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms. 

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5. 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 moiety of BR leads to high flexibility and structural heterogeneity of UnaG N57A, as seen in the X-ray crystal structure. 

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