Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.
International journal of molecular sciences
2023; 24 (15)
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.
View details for DOI 10.3390/ijms241511991
View details for PubMedID 37569365
Protein protic and aprotic interactions systematically mapped via IR spectroscopy and polarizable molecular dynamics
CELL PRESS. 2023: 309A
View details for Web of Science ID 000989629701625
- Protein protic and aprotic interactions systematically mapped via IR spectroscopy and polarizable molecular dynamics. Biophysical journal 2023; 122 (3S1): 309a
- Carbon-deuterium bonds as reporters of electric fields in solvent and protein environments. Biophysical journal 2023; 122 (3S1): 481a
Carbon-deuterium bonds as reporters of electric fields in solvent and protein environments
CELL PRESS. 2023: 481A
View details for Web of Science ID 000989629702586
Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments.
Journal of the American Chemical Society
Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the electric field projected on the -C=N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to both H-bonding and non-H-bonding interactions. It can therefore be generally applied to determine electric fields in all environments. Additionally, it allows for semiempirical extraction of the H-bonding contribution to the blueshift of the nitrile frequency. Nitriles were incorporated at H-bonding and non-H-bonding protein sites using amber suppression, and each nitrile variant was structurally characterized at high resolution. We exploited the combined information available from variations in frequency and integrated intensity and demonstrate that nitriles are a generally useful probe for electric fields.
View details for DOI 10.1021/jacs.2c00675
View details for PubMedID 35467853
Nitrile IR intensities directly measure electric fields in protic and non-protic environments
CELL PRESS. 2022: 414A
View details for Web of Science ID 000759523002558