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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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Directed evolution of a far-red fluorescent rhodopsin.

R Scott McIsaac¹, Martin K M Engqvist¹, Timothy Wannier²

¹Divisions of Chemistry and Chemical Engineering and.

Proceedings of the National Academy of Sciences of the United States of America

|August 27, 2014

Summary

Researchers enhanced the brightness of microbial rhodopsins (Arch) for improved fluorescent imaging. These brightened Arch variants are useful for labeling biological membranes and voltage sensing in live cells.

Keywords:

bioelectricity near-infrared opsins optogenetics voltage sensor

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Area of Science:

Molecular Biology
Biophysics
Microbiology

Background:

Microbial rhodopsins are photoactive transmembrane proteins present in all domains of life.
Archaerhodopsin-3 (Arch) from Halobacterium Halorubrum sodomense functions as a fluorescent membrane potential indicator.
Native Arch exhibits low fluorescence, limiting its utility in live-cell imaging applications.

Purpose of the Study:

To improve the absolute brightness of Archaerhodopsin-3 (Arch) for enhanced live-cell imaging.
To develop novel fluorescent protein variants for membrane potential sensing and biological membrane labeling.

Main Methods:

Employed directed evolution techniques to introduce mutations into Arch.
Biochemical assays and live-cell imaging in *Escherichia coli* and human embryonic kidney 293 cells were used for characterization.
Analyzed fluorescence properties, including pK(a) of the Schiff-base linkage and excitation/emission spectra.

Main Results:

Identified Arch variants with dramatically improved absolute brightness compared to the wild-type.
Some variants displayed a pK(a) near neutral pH, suitable for voltage-sensing.
Achieved far-red/infrared fluorescence emission (max ~620 nm/730 nm), the furthest red-shifted reported for fluorescent proteins.

Conclusions:

Directed evolution successfully generated significantly brighter Arch variants.
These enhanced Arch proteins are valuable tools for live-cell imaging, membrane labeling, and voltage sensing.
The far-red/infrared emission expands the spectral range for fluorescent protein applications.