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Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

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Structure-guided engineering enhances a phytochrome-based infrared fluorescent protein.

Michele E Auldridge¹, Kenneth A Satyshur, David M Anstrom

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.

The Journal of Biological Chemistry

|January 3, 2012

Summary

Researchers engineered a brighter infrared fluorescent protein from phytochrome. This Wisconsin infrared phytofluor (Wi-Phy) variant is smaller and suitable for in vivo imaging applications.

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Published on: May 29, 2010

Area of Science:

Biochemistry
Biophysics
Molecular Biology

Background:

Phytochromes are red light photoreceptors crucial for plant development.
A modified phytochrome chromophore-binding domain (CBD) shows potential as an infrared fluorophore.
Understanding structural determinants of fluorescence is key for biotechnological applications.

Purpose of the Study:

To characterize structural and functional properties of engineered phytochrome CBD variants.
To develop a brighter and smaller infrared fluorescent protein for in vivo applications.
To investigate the role of dimerization in phytochrome photochemistry.

Main Methods:

X-ray crystallography was used to determine the structures of D207H and Y263F variants.
Site-directed mutagenesis was employed to create monomeric and fluorescent variants.
Fluorescence properties and photoconversion were analyzed.

Main Results:

Crystal structures revealed His-207 hydrogen bonding duality, impacting fluorescence.
The Y263F substitution enhanced fluorescence by preventing red-shifted states.
A monomeric CBD variant (Wi-Phy) exhibited enhanced brightness and unexpected photoconversion.
Wi-Phy, combining D207H and Y263F in a monomer, showed the strongest fluorescence.

Conclusions:

Structural modifications of phytochrome CBD can yield highly fluorescent infrared probes.
The Y263F mutation is critical for bright fluorescence by stabilizing a specific chromophore configuration.
Monomeric phytochrome CBD variants offer new avenues for studying photochemistry and can serve as advanced fluorescent tags.