Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment

The design of fluorescent protein (FP) mutants with tailored properties benefits from the comprehension of chromophore structure, interactions, energy landscapes, and dynamics. Vibrational spectroscopy can often provide detailed information on these characteristics for proteins in their natural aqueous environment, during their (photo)dynamics and without the need of crystallization. Here we will review the experimental and theoretical techniques that have been used to analyze the relations between vibrational spectra and different structural, photophysical, and chemical properties of FPs, in particular the ones able to selectively address the chromophore and its close environment, like (pre)resonance Raman, difference-IR absorption measurements, and their computational simulations. Starting from the preliminary results aimed at identifying vibrational modes in the neutral and anionic GFP chromophore, we will discuss selected experiments that unraveled, often thanks to comparison with theoretical studies, the structure of the chromophore in some FP mutants, the impact of cis\u2013trans isomerization and different protonation states in reversibly photoswitchable proteins, and the structural changes and proton-transfer pathway in the photoexcited state