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Related Experiment Video

Updated: Jun 2, 2026

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Published on: June 26, 2010

Decarboxylation via a Higher Electronic Excited State Drives LSSmOrange Photoconversion.

Hyang Sook Seol¹, Fangjia Luo², Elke De Zitter³

¹Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Celestijnenlaan 200G bus 2403, Leuven 3001, Belgium.

ACS Physical Chemistry Au

|June 1, 2026

Summary

LSSmOrange photoconversion involves multiphoton absorption, leading to spatially confined highlighting. This study used serial femtosecond crystallography to reveal the excited state dynamics and structural changes during this process.

Keywords:

Kolbe decarboxylation X-ray free electron laser photoswitchable fluorescent proteins time-resolved serial femtosecond crystallography time-resolved spectroscopy

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Area of Science:

Biophysics
Structural Biology
Photochemistry

Background:

LSSmOrange is a fluorescent protein used for molecular imaging.
Its photoconversion mechanism, involving Kolbe decarboxylation at E215, is known but its structural dynamics are not.
Understanding these dynamics is crucial for optimizing its use as an imaging tool.

Purpose of the Study:

To investigate the excited state dynamics and time-resolved structural changes of LSSmOrange during photoconversion.
To elucidate the mechanism underlying LSSmOrange photoconversion, particularly given its low single-photon quantum yield.

Main Methods:

Serial femtosecond crystallography (SFX) with an X-ray free electron laser to determine high-resolution structures.
Femtosecond optical pump–X-ray probe experiments to track time-resolved structural changes.
Transient absorption spectroscopy and quantum-chemical calculations to investigate the photoconversion mechanism.

Main Results:

SFX provided damage-free structures of unconverted LSSmOrange.
A decrease in electron density at E215 was observed 250 ps post-illumination, confirming decarboxylation.
Evidence suggests photoconversion proceeds via a multiphoton absorption process involving a higher-lying electronic excited state, linked to optical nonlinearity.

Conclusions:

LSSmOrange photoconversion is driven by multiphoton absorption, not single-photon absorption, explaining its efficiency despite a low quantum yield.
This multiphoton mechanism allows for spatially confined highlighting, enhancing its utility in microscopy.
The study reveals novel insights into the excited state dynamics and structural rearrangements of LSSmOrange.