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

Cheng Chen1, J Nathan Henderson2, Dmitry A Ruchkin3

  • 1Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331, USA.

International Journal of Molecular Sciences
|August 12, 2023
PubMed
Summary
This summary is machine-generated.

Wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) reveals conserved vibrational markers in fluorescent proteins (FPs). This technique elucidates chromophore-environment interactions, aiding in the engineering of FPs for diverse applications.

Keywords:
chromophore–environment interactionscis and trans conformationsfemtosecond stimulated Raman spectroscopygreen fluorescent proteinred fluorescent proteinresonance structures

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Area of Science:

  • Biophysical Chemistry
  • Spectroscopy
  • Protein Engineering

Background:

  • Fluorescent proteins (FPs) are vital fluorescence biomarkers whose functions rely on chromophore-environment interactions.
  • Vibrational spectroscopy, alongside X-ray crystallography, offers critical insights into chromophore structure and interactions.

Purpose of the Study:

  • To benchmark ground-state vibrational signatures of diverse FPs and their model chromophores.
  • To analyze factors influencing FP chromophore vibrational properties, including structure and environment.
  • To establish wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) as a powerful tool for FP characterization.

Main Methods:

  • Utilized wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS).
  • Investigated a series of FPs emitting across green to red wavelengths, plus solvated model chromophores.
  • Employed quantum calculations to complement spectroscopic data analysis.

Main Results:

  • Identified a conserved bond-stretching mode characteristic of the quinoidal resonance structure in most FPs and model chromophores.
  • Demonstrated that this mode serves as a vibrational marker for interpreting chromophore-environment interactions and electronic property effects.
  • Showcased the high sensitivity and resolution of FSRS in analyzing FP chromophores in solution and protein matrices.

Conclusions:

  • Fundamental insights into FP chromophore properties were gained using wavelength-tunable FSRS.
  • The conserved vibrational marker facilitates understanding of structural effects on chromophore electronic properties.
  • This comprehensive characterization provides a foundation for future spectroscopic studies and rational FP engineering.