Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman...

Area of Science:

• Photochemistry and Photophysics
• Biophysical Chemistry
• Spectroscopy

Background:

• Tracking molecular motions is crucial for understanding photochemical processes.
• Engineered green fluorescent proteins (GFPs) offer tunable fluorescence properties.
• Excited state proton transfer (ESPT) is a key mechanism in many fluorescent proteins.

Purpose of the Study:

• To monitor the excited state structural evolution of an engineered GFP single-site mutant (S205V) using ultrafast vibrational spectroscopy.
• To elucidate the role of specific vibrational modes and the rate-limiting step in the ESPT process.
• To gain insights into the fluorescence mechanisms of engineered GFPs.

Main Methods:

• Femtosecond-stimulated Raman spectroscopy (FSRS) was employed to track ultrafast dynamics.
• Selective monitoring of excited protonated (A*) and deprotonated (I*) species was achieved by tuning the Raman pump wavelength.
• Analysis of characteristic Raman modes and their time-dependent evolution provided structural information.

Main Results:

• The rate-limiting step for ESPT in the S205V GFP mutant was identified as the bridging of a water molecule to protein residue T203, occurring on a ~300 ps timescale.
• Vibrational cooling of A* species was observed on ~190 ps and ~80 ps timescales.
• A ~1330 cm-1 delocalized motion with dispersive line shapes indicated strong vibronic coupling, facilitating the transition to a stable state.

Conclusions:

• The study successfully disentangled the contributions of various vibrational motions during the ESPT reaction in engineered GFP.
• Findings provide new structural dynamics insights into the fluorescence mechanisms of engineered GFPs and related autofluorescent proteins.
• Femtosecond-stimulated Raman spectroscopy is a powerful tool for investigating ultrafast dynamics in complex biological systems.