Incorporation of sensing modalities into de novo designed fluorescence-activating proteins

Jason C. Klima, Lindsey A. Doyle, Justin Daho Lee, Michael Rappleye, Lauren A. Gagnon, Min Yen Lee, Emilia P. Barros, Anastassia A. Vorobieva, Jiayi Dou, Samantha Bremner, Jacob S. Quon, Cameron M. Chow, Lauren Carter, David L. Mack, Rommie E. Amaro, Joshua C. Vaughan, Andre Berndt, Barry L. Stoddard and David Baker ()
Additional contact information
Jason C. Klima: University of Washington
Lindsey A. Doyle: Fred Hutchinson Cancer Research Center
Justin Daho Lee: University of Washington
Michael Rappleye: University of Washington
Lauren A. Gagnon: University of Washington
Min Yen Lee: University of Washington
Emilia P. Barros: University of California, San Diego
Anastassia A. Vorobieva: University of Washington
Jiayi Dou: University of Washington
Samantha Bremner: University of Washington
Jacob S. Quon: University of Washington
Cameron M. Chow: University of Washington
Lauren Carter: University of Washington
David L. Mack: University of Washington
Rommie E. Amaro: University of California, San Diego
Joshua C. Vaughan: University of Washington
Andre Berndt: University of Washington
Barry L. Stoddard: Fred Hutchinson Cancer Research Center
David Baker: University of Washington

Nature Communications, 2021, vol. 12, issue 1, 1-19

Abstract: Abstract Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein–protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

Date: 2021
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DOI: 10.1038/s41467-020-18911-w

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