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:
-
- 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}
}
Works referenced in this record:
Distinct Metabolic Flow Enables Large-Scale Purification of Mouse and Human Pluripotent Stem Cell-Derived Cardiomyocytes
journal, January 2013
- Tohyama, Shugo; Hattori, Fumiyuki; Sano, Motoaki
- Cell Stem Cell, Vol. 12, Issue 1
Bright and photostable chemigenetic indicators for extended in vivo voltage imaging
journal, August 2019
- Abdelfattah, Ahmed S.; Kawashima, Takashi; Singh, Amrita
- Science, Vol. 365, Issue 6454
Calorimetric determinations of absolute fluorescence quantum yields
journal, October 1979
- Olmsted, John.
- The Journal of Physical Chemistry, Vol. 83, Issue 20
-Helix nucleation by a calcium-binding peptide loop
journal, February 1999
- Siedlecka, M.; Goch, G.; Ejchart, A.
- Proceedings of the National Academy of Sciences, Vol. 96, Issue 3
R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes
journal, July 2016
- Bovo, Elisa; Martin, Jody L.; Tyryfter, Jollyn
- American Journal of Physiology-Heart and Circulatory Physiology, Vol. 311, Issue 1
Calcium Affinity, Cooperativity, and Domain Interactions of Extracellular EF-hands Present in BM-40
journal, August 2000
- Busch, Elisabeth; Hohenester, Erhard; Timpl, Rupert
- Journal of Biological Chemistry, Vol. 275, Issue 33
Ultrasensitive fluorescent proteins for imaging neuronal activity
journal, July 2013
- Chen, Tsai-Wen; Wardill, Trevor J.; Sun, Yi
- Nature, Vol. 499, Issue 7458
Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System
journal, May 2019
- Smith, Elizabeth M.; Gautier, Arnaud; Puchner, Elias M.
- ACS Chemical Biology, Vol. 14, Issue 6
Improved split fluorescent proteins for endogenous protein labeling
journal, August 2017
- Feng, Siyu; Sekine, Sayaka; Pessino, Veronica
- Nature Communications, Vol. 8, Issue 1
Automatic atom type and bond type perception in molecular mechanical calculations
journal, October 2006
- Wang, Junmei; Wang, Wei; Kollman, Peter A.
- Journal of Molecular Graphics and Modelling, Vol. 25, Issue 2
Towards automated crystallographic structure refinement with phenix.refine
journal, March 2012
- Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
- Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics
journal, May 2019
- Inoue, Masatoshi; Takeuchi, Atsuya; Manita, Satoshi
- Cell, Vol. 177, Issue 5
SwiftLib: rapid degenerate-codon-library optimization through dynamic programming
journal, December 2014
- Jacobs, Timothy M.; Yumerefendi, Hayretin; Kuhlman, Brian
- Nucleic Acids Research, Vol. 43, Issue 5
Relative and absolute determination of fluorescence quantum yields of transparent samples
journal, July 2013
- Würth, Christian; Grabolle, Markus; Pauli, Jutta
- Nature Protocols, Vol. 8, Issue 8
Comparison of simple potential functions for simulating liquid water
journal, July 1983
- Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.
- The Journal of Chemical Physics, Vol. 79, Issue 2
Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs. 2. Explicit Solvent Particle Mesh Ewald
journal, August 2013
- Salomon-Ferrer, Romelia; Götz, Andreas W.; Poole, Duncan
- Journal of Chemical Theory and Computation, Vol. 9, Issue 9
An allosteric model of calmodulin explains differential activation of PP2B and CaMKII
journal, July 2008
- Stefan, M. I.; Edelstein, S. J.; Le Novere, N.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 31
Protein Allostery and Conformational Dynamics
journal, February 2016
- Guo, Jingjing; Zhou, Huan-Xiang
- Chemical Reviews, Vol. 116, Issue 11
Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm
journal, April 2016
- Li, ZhaoYang; Zhang, ZhiPing; Bi, LiJun
- PLOS ONE, Vol. 11, Issue 4
Structure of Escherichia coli fragment TR 2 C from calmodulin to 1.7 Å resolution
journal, April 2001
- Olsson, Lise-Lotte; Sjölin, Lennart
- Acta Crystallographica Section D Biological Crystallography, Vol. 57, Issue 5
Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex
journal, September 2017
- Song, Wenjiao; Filonov, Grigory S.; Kim, Hyaeyeong
- Nature Chemical Biology, Vol. 13, Issue 11
Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy
journal, December 2015
- Liss, Viktoria; Barlag, Britta; Nietschke, Monika
- Scientific Reports, Vol. 5, Issue 1
pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein
journal, January 2011
- Mahon, Matthew John
- Advances in Bioscience and Biotechnology, Vol. 02, Issue 03
Development and testing of a general amber force field
journal, January 2004
- Wang, Junmei; Wolf, Romain M.; Caldwell, James W.
- Journal of Computational Chemistry, Vol. 25, Issue 9
Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators
journal, November 2009
- Tian, Lin; Hires, S Andrew; Mao, Tianyi
- Nature Methods, Vol. 6, Issue 12, p. 875-881
Imaging Intracellular pH in Live Cells with a Genetically Encoded Red Fluorescent Protein Sensor
journal, July 2011
- Tantama, Mathew; Hung, Yin Pun; Yellen, Gary
- Journal of the American Chemical Society, Vol. 133, Issue 26
X-Ray and molecular dynamics studies of concanavalin-A glucoside and mannoside complexes Relating structure to thermodynamics of binding
journal, January 1998
- Bradbrook, Gail M.; Gleichmann, Thomas; Harrop, Stephen J.
- Journal of the Chemical Society, Faraday Transactions, Vol. 94, Issue 11
PyEMMA 2: A Software Package for Estimation, Validation, and Analysis of Markov Models
journal, October 2015
- Scherer, Martin K.; Trendelkamp-Schroer, Benjamin; Paul, Fabian
- Journal of Chemical Theory and Computation, Vol. 11, Issue 11
RosettaRemodel: A Generalized Framework for Flexible Backbone Protein Design
journal, August 2011
- Huang, Po-Ssu; Ban, Yih-En Andrew; Richter, Florian
- PLoS ONE, Vol. 6, Issue 8
The Rosetta All-Atom Energy Function for Macromolecular Modeling and Design
journal, May 2017
- Alford, Rebecca F.; Leaver-Fay, Andrew; Jeliazkov, Jeliazko R.
- Journal of Chemical Theory and Computation, Vol. 13, Issue 6
MDTraj: A Modern Open Library for the Analysis of Molecular Dynamics Trajectories
journal, October 2015
- McGibbon, Robert T.; Beauchamp, Kyle A.; Harrigan, Matthew P.
- Biophysical Journal, Vol. 109, Issue 8
Split-miniSOG for Spatially Detecting Intracellular Protein-Protein Interactions by Correlated Light and Electron Microscopy
journal, October 2019
- Boassa, Daniela; Lemieux, Sakina P.; Lev-Ram, Varda
- Cell Chemical Biology, Vol. 26, Issue 10
The fluorescent protein palette: tools for cellular imaging
journal, January 2009
- Day, Richard N.; Davidson, Michael W.
- Chemical Society Reviews, Vol. 38, Issue 10
A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association
journal, October 2013
- Cabantous, Stéphanie; Nguyen, Hau B.; Pedelacq, Jean-Denis
- Scientific Reports, Vol. 3, Issue 1
Control over overall shape and size in de novo designed proteins
journal, September 2015
- Lin, Yu-Ru; Koga, Nobuyasu; Tatsumi-Koga, Rie
- Proceedings of the National Academy of Sciences, Vol. 112, Issue 40
Imaging dynamic cell signaling in vivo with new classes of fluorescent reporters
journal, February 2020
- Shu, Xiaokun
- Current Opinion in Chemical Biology, Vol. 54
Bimolecular Fluorescence Complementation (BiFC) Analysis as a Probe of Protein Interactions in Living Cells
journal, June 2008
- Kerppola, Tom K.
- Annual Review of Biophysics, Vol. 37, Issue 1
Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach
journal, January 2014
- Song, Wenjiao; Strack, Rita L.; Svensen, Nina
- Journal of the American Chemical Society, Vol. 136, Issue 4
Absorption and fluorescence properties of fluorescein
journal, June 1995
- Sjöback, Robert; Nygren, Jan; Kubista, Mikael
- Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, Vol. 51, Issue 6
ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
journal, July 2015
- Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik
- Journal of Chemical Theory and Computation, Vol. 11, Issue 8
Features and development of Coot
journal, March 2010
- Emsley, P.; Lohkamp, B.; Scott, W. G.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
Macromolecular Modeling with Rosetta
journal, June 2008
- Das, Rhiju; Baker, David
- Annual Review of Biochemistry, Vol. 77, Issue 1
Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer
journal, November 2016
- Berger, Stephanie; Procko, Erik; Margineantu, Daciana
- eLife, Vol. 5
Circularly permuted green fluorescent proteins engineered to sense Ca2+
journal, March 2001
- Nagai, T.; Sawano, A.; Park, E. S.
- Proceedings of the National Academy of Sciences, Vol. 98, Issue 6, p. 3197-3202
Cyclopiazonic Acid is a Specific Inhibitor of the Ca2+-ATPase of Sarcoplasmic Reticulum
journal, October 1989
- Seidler, N. W.; Jona, I.; Vegh, M.
- Journal of Biological Chemistry, Vol. 264, Issue 30
Fiji: an open-source platform for biological-image analysis
journal, June 2012
- Schindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin
- Nature Methods, Vol. 9, Issue 7
Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization
journal, May 2013
- Skinner, Samuel O.; Sepúlveda, Leonardo A.; Xu, Heng
- Nature Protocols, Vol. 8, Issue 6
PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
A Closed Compact Structure of Native Ca2+-Calmodulin
journal, October 2003
- Fallon, Jennifer L.; Quiocho, Florante A.
- Structure, Vol. 11, Issue 10
A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store
journal, October 2015
- Henderson, Mark J.; Baldwin, Heather A.; Werley, Christopher A.
- PLOS ONE, Vol. 10, Issue 10
Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments
journal, September 2011
- Tang, S.; Wong, H. -C.; Wang, Z. -M.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 39
iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications
journal, March 2016
- Autour, Alexis; Westhof, Eric; Ryckelynck, Michael
- Nucleic Acids Research, Vol. 44, Issue 6
Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo
journal, December 2015
- Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie
- Proceedings of the National Academy of Sciences, Vol. 113, Issue 3
New approaches for sensing metabolites and proteins in live cells using RNA
journal, August 2013
- Strack, Rita L.; Jaffrey, Samie R.
- Current Opinion in Chemical Biology, Vol. 17, Issue 4
Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors
journal, August 2018
- Tebo, Alison G.; Pimenta, Frederico M.; Zoumpoulaki, Martha
- ACS Chemical Biology, Vol. 13, Issue 9
HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis
journal, June 2008
- Los, Georgyi V.; Encell, Lance P.; McDougall, Mark G.
- ACS Chemical Biology, Vol. 3, Issue 6
Circular permutation and receptor insertion within green fluorescent proteins
journal, September 1999
- Baird, G. S.; Zacharias, D. A.; Tsien, R. Y.
- Proceedings of the National Academy of Sciences, Vol. 96, Issue 20
Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions
journal, December 2012
- Lian, Xiaojun; Zhang, Jianhua; Azarin, Samira M.
- Nature Protocols, Vol. 8, Issue 1
A split fluorescent reporter with rapid and reversible complementation
journal, June 2019
- Tebo, Alison G.; Gautier, Arnaud
- Nature Communications, Vol. 10, Issue 1
RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite
journal, June 2011
- Fleishman, Sarel J.; Leaver-Fay, Andrew; Corn, Jacob E.
- PLoS ONE, Vol. 6, Issue 6
Regulation of the Protein-Conducting Channel by a Bound Ribosome
journal, November 2009
- Gumbart, James; Trabuco, Leonardo G.; Schreiner, Eduard
- Structure, Vol. 17, Issue 11
RNA Mimics of Green Fluorescent Protein
journal, July 2011
- Paige, J. S.; Wu, K. Y.; Jaffrey, S. R.
- Science, Vol. 333, Issue 6042
An improved inverse-type Ca2+ indicator can detect putative neuronal inhibition in Caenorhabditis elegans by increasing signal intensity upon Ca2+ decrease
journal, April 2018
- Hara-Kuge, Sayuri; Nishihara, Tomonobu; Matsuda, Tomoki
- PLOS ONE, Vol. 13, Issue 4
Localization Microscopy using Noncovalent Fluorogen Activation by Genetically Encoded Fluorogen-Activating Proteins.
journal, November 2013
- Yan, Qi; Schwartz, Samantha L.; Maji, Suvrajit
- ChemPhysChem, Vol. 15, Issue 4
Phaser crystallographic software
journal, July 2007
- McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
- Journal of Applied Crystallography, Vol. 40, Issue 4
VMD: Visual molecular dynamics
journal, February 1996
- Humphrey, William; Dalke, Andrew; Schulten, Klaus
- Journal of Molecular Graphics, Vol. 14, Issue 1
De novo design of a fluorescence-activating β-barrel
journal, September 2018
- Dou, Jiayi; Vorobieva, Anastassia A.; Sheffler, William
- Nature, Vol. 561, Issue 7724
Predicting protein structures with a multiplayer online game
journal, August 2010
- Cooper, Seth; Khatib, Firas; Treuille, Adrien
- Nature, Vol. 466, Issue 7307
PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data
journal, June 2013
- Roe, Daniel R.; Cheatham, Thomas E.
- Journal of Chemical Theory and Computation, Vol. 9, Issue 7