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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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This content will become publicly available on August 12, 2026
A twisted chromophore powers a turn-on fluorescent protein chloride sensor
Fluorescent proteins (FPs) are noninvasive genetically encodable probes that have revolutionized bioimaging and health fields with vivid images and an ever-growing repertoire from jellyfish to sea anemones and corals. Inside the protein matrix, chromophore nonplanarity and flexibility have long been argued to govern the fluorescence efficiency of FPs, yet their fundamental roles and relative importance have been elusive which hinder the rational design of versatile FPs and biosensors. Herein, we tackle this central question by investigating two recently engineered FP-based turn-on chloride (Cl–) sensors, ChlorON1 and 3, using an ultrafast electronic and vibrational spectroscopic toolset together with advanced multireference simulations for both structure and spectrum. We elucidate that fluorescence enhancement of the chloride-bound ChlorON3 stems from a substantially more twisted chromophore than ChlorON1 via comprehensive simulations starting from the available crystal structure of parent protein (mNeonGreen), also featuring an enhanced radiative pathway due to an adjacent leucine residue in the emissive population. This finding indicates that the commonly stated chromophore planarity is not, but conformational rigidity is, the decisive factor for high fluorescence efficiency. Such mechanistic insights into FPs are generalizable to chromoproteins and other photosensitive biomolecules, which can facilitate the targeted design of brighter and/or tunable biosensors.
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- PAR ID:
- 10627856
- Publisher / Repository:
- PNAS
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 122
- Issue:
- 32
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- e2508094122
- Subject(s) / Keyword(s):
- fluorescent protein-based biosensors structural dynamics rigidity control ultrafast laser spectroscopy rational protein design BIOPHYSICS AND COMPUTATIONAL BIOLOGY CHEMISTRY
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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