Incorporation of sensing modalities into de novo designed fluorescence-activating proteins
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 fluorescentcis-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.
- Authors:
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[1];
[2];
[3];
[4];
[5];
[1];
[4];
[7];
[1];
[6];
[9];
[10]
- Univ. of Washington, Seattle, WA (United States). Dept. of Biochemistry; Univ. of Washington, Seattle, WA (United States). Inst. for Protein Design
- Fred Hutchinson Cancer Research Center, Seattle, WA (United States). Division of Basic Sciences
- Univ. of Washington, Seattle, WA (United States). Molecular Engineering Ph.D. Program; Univ. of Washington, Seattle, WA (United States). Inst. for Stem Cell and Regenerative Medicine; Univ. of Washington, Seattle, WA (United States). Dept. of Bioengineering
- Univ. of Washington, Seattle, WA (United States). Inst. for Stem Cell and Regenerative Medicine; Univ. of Washington, Seattle, WA (United States). Dept. of Bioengineering
- Univ. of Washington, Seattle, WA (United States). Dept. of Chemistry
- Univ. of California, San Diego, La Jolla, CA (United States). Dept. of Chemistry and Biochemistry
- Univ. of Washington, Seattle, WA (United States). Inst. for Protein Design
- Univ. of Washington, Seattle, WA (United States). Inst. for Stem Cell and Regenerative Medicine; Univ. of Washington, Seattle, WA (United States). Dept. of Bioengineering; Univ. of Washington, Seattle, WA (United States). Dept. of Rehabilitation Medicine
- Univ. of Washington, Seattle, WA (United States). Dept. of Chemistry; Univ. of Washington, Seattle, WA (United States). Dept. of Physiology and Biophysics
- Univ. of Washington, Seattle, WA (United States). Dept. of Biochemistry; Univ. of Washington, Seattle, WA (United States). Inst. for Protein Design; Univ. of Washington, Seattle, WA (United States). Molecular Engineering Ph.D. Program; Univ. of Washington, Seattle, WA (United States). Howard Hughes Medical Inst.
- Publication Date:
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; National Science Foundation (NSF)
- OSTI Identifier:
- 1817045
- Grant/Contract Number:
- AC02-05CH11231; DGE-1256082; NNCI-1542101
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; fluorescent proteins; protein design; wide-field fluorescence microscopy; x-ray crystallography
Citation Formats
Klima, Jason C., Doyle, Lindsey A., Lee, Justin Daho, Rappleye, Michael, Gagnon, Lauren A., Lee, Min Yen, Barros, Emilia P., Vorobieva, Anastassia A., Dou, Jiayi, Bremner, Samantha, Quon, Jacob S., Chow, Cameron M., Carter, Lauren, Mack, David L., Amaro, Rommie E., Vaughan, Joshua C., Berndt, Andre, Stoddard, Barry L., and Baker, David. Incorporation of sensing modalities into de novo designed fluorescence-activating proteins. United States: N. p., 2021.
Web. doi:10.1038/s41467-020-18911-w.
Klima, Jason C., Doyle, Lindsey A., Lee, Justin Daho, Rappleye, Michael, Gagnon, Lauren A., Lee, Min Yen, Barros, Emilia P., Vorobieva, Anastassia A., Dou, Jiayi, Bremner, Samantha, Quon, Jacob S., Chow, Cameron M., Carter, Lauren, Mack, David L., Amaro, Rommie E., Vaughan, Joshua C., Berndt, Andre, Stoddard, Barry L., & Baker, David. Incorporation of sensing modalities into de novo designed fluorescence-activating proteins. United States. https://doi.org/10.1038/s41467-020-18911-w
Klima, Jason C., Doyle, Lindsey A., Lee, Justin Daho, Rappleye, Michael, Gagnon, Lauren A., Lee, Min Yen, Barros, Emilia P., Vorobieva, Anastassia A., Dou, Jiayi, Bremner, Samantha, Quon, Jacob S., Chow, Cameron M., Carter, Lauren, Mack, David L., Amaro, Rommie E., Vaughan, Joshua C., Berndt, Andre, Stoddard, Barry L., and Baker, David. Mon .
"Incorporation of sensing modalities into de novo designed fluorescence-activating proteins". United States. https://doi.org/10.1038/s41467-020-18911-w. https://www.osti.gov/servlets/purl/1817045.
@article{osti_1817045,
title = {Incorporation of sensing modalities into de novo designed fluorescence-activating proteins},
author = {Klima, Jason C. and Doyle, Lindsey A. and Lee, Justin Daho and Rappleye, Michael and Gagnon, Lauren A. and Lee, Min Yen and Barros, Emilia P. and Vorobieva, Anastassia A. and Dou, Jiayi and Bremner, Samantha and Quon, Jacob S. and Chow, Cameron M. and Carter, Lauren and Mack, David L. and Amaro, Rommie E. and Vaughan, Joshua C. and Berndt, Andre and Stoddard, Barry L. and Baker, David},
abstractNote = {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 fluorescentcis-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.},
doi = {10.1038/s41467-020-18911-w},
journal = {Nature Communications},
number = 1,
volume = 12,
place = {United States},
year = {Mon Feb 08 00:00:00 EST 2021},
month = {Mon Feb 08 00:00:00 EST 2021}
}
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