Fluorescence Modulation by Ultrafast Chromophore Twisting Events: Developing a Powerful Toolset for...

Area of Science:

• Biophysics
• Spectroscopy
• Protein Engineering

Background:

• Fluorescent proteins (FPs) are vital tools in life sciences for tracking cellular events.
• FP chromophores exhibit ultrafast photophysical processes but lose fluorescence in solution due to photoexcitation-induced twisting.
• Understanding these microscopic torsional events is key to controlling macroscopic fluorescence.

Purpose of the Study:

• To investigate the role of torsional motions in the photophysics of various fluorescent proteins.
• To develop and apply an integrated ultrafast spectroscopy platform for detailed FP characterization.
• To correlate electronic and structural dynamics with FP fluorescence properties for rational design.

Main Methods:

• Developed an integrated ultrafast characterization platform combining femtosecond transient absorption (fs-TA) and wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS).
• Investigated a diverse range of FPs, including naturally occurring, circularly permuted, and engineered variants with optical highlighters.
• Monitored ultrafast pathways by analyzing frequency changes of characteristic Raman bands during primary photophysical events.

Main Results:

• Twisting conformational motions were observed in all investigated FP systems, varying in extent.
• Ultrafast dynamics and associated pathways were successfully monitored using the integrated spectroscopy platform.
• Detailed electronic and structural dynamics information was mapped for various FPs and FP-based tools.

Conclusions:

• The study elucidates the significant role of twisting motions in FP photophysics across a wide range of engineered proteins.
• The developed ultrafast spectroscopy platform provides crucial insights into the dynamics of FPs.
• This research shows great potential for the rational design of novel photoreceptors and fluorescent proteins with tailored functions and improved fluorescence properties.