Development of an optical Zn2+ probe based on a single fluorescent protein
Abstract
Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1 which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). In conclusion, this sensor design provides freedommore »
- Authors:
-
- Univ. of Colorado, Boulder, CO (United States)
- National Renewable Energy Lab. (NREL), Golden, CO (United States)
- Publication Date:
- Research Org.:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
- OSTI Identifier:
- 1330942
- Report Number(s):
- NREL/JA-2700-66894
Journal ID: ISSN 1554-8929
- Grant/Contract Number:
- AC36-08GO28308
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ACS Chemical Biology
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 10; Journal ID: ISSN 1554-8929
- Publisher:
- American Chemical Society
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; zinc; genetically encoded sensor; single fluorescent protein; Rosetta
Citation Formats
Qin, Yan, Sammond, Deanne W., Braselmann, Esther, Carpenter, Margaret C., and Palmer, Amy E. Development of an optical Zn2+ probe based on a single fluorescent protein. United States: N. p., 2016.
Web. doi:10.1021/acschembio.6b00442.
Qin, Yan, Sammond, Deanne W., Braselmann, Esther, Carpenter, Margaret C., & Palmer, Amy E. Development of an optical Zn2+ probe based on a single fluorescent protein. United States. https://doi.org/10.1021/acschembio.6b00442
Qin, Yan, Sammond, Deanne W., Braselmann, Esther, Carpenter, Margaret C., and Palmer, Amy E. Thu .
"Development of an optical Zn2+ probe based on a single fluorescent protein". United States. https://doi.org/10.1021/acschembio.6b00442. https://www.osti.gov/servlets/purl/1330942.
@article{osti_1330942,
title = {Development of an optical Zn2+ probe based on a single fluorescent protein},
author = {Qin, Yan and Sammond, Deanne W. and Braselmann, Esther and Carpenter, Margaret C. and Palmer, Amy E.},
abstractNote = {Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1 which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). In conclusion, this sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.},
doi = {10.1021/acschembio.6b00442},
journal = {ACS Chemical Biology},
number = 10,
volume = 11,
place = {United States},
year = {2016},
month = {7}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 27 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Visualizing metal ions in cells: An overview of analytical techniques, approaches, and probes
journal, September 2012
- Dean, Kevin M.; Qin, Yan; Palmer, Amy E.
- Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1823, Issue 9
Metals in Neurobiology: Probing Their Chemistry and Biology with Molecular Imaging
journal, May 2008
- Que, Emily L.; Domaille, Dylan W.; Chang, Christopher J.
- Chemical Reviews, Vol. 108, Issue 5
Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design
journal, December 2008
- Akerboom, Jasper; Rivera, Jonathan D. Vélez; Guilbe, María M. Rodríguez
- Journal of Biological Chemistry, Vol. 284, Issue 10
Direct Comparison of a Genetically Encoded Sensor and Small Molecule Indicator: Implications for Quantification of Cytosolic Zn 2+
journal, September 2013
- Qin, Yan; Miranda, Jose G.; Stoddard, Caitlin I.
- ACS Chemical Biology, Vol. 8, Issue 11
Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis
journal, August 2009
- Vinkenborg, Jan L.; Nicolson, Tamara J.; Bellomo, Elisa A.
- Nature Methods, Vol. 6, Issue 10
Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors
journal, April 2011
- Qin, Y.; Dittmer, P. J.; Park, J. G.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 18
Fluorescent Sensors for Measuring Metal Ions in Living Systems
journal, March 2014
- Carter, Kyle P.; Young, Alexandra M.; Palmer, Amy E.
- Chemical Reviews, Vol. 114, Issue 8
New Sensors for Quantitative Measurement of Mitochondrial Zn 2+
journal, August 2012
- Park, J. Genevieve; Qin, Yan; Galati, Domenico F.
- ACS Chemical Biology, Vol. 7, Issue 10
Mitochondrial and ER-Targeted eCALWY Probes Reveal High Levels of Free Zn 2+
journal, July 2014
- Chabosseau, Pauline; Tuncay, Erkan; Meur, Gargi
- ACS Chemical Biology, Vol. 9, Issue 9
eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn 2+ Imaging
journal, July 2015
- Hessels, Anne M.; Chabosseau, Pauline; Bakker, Maarten H.
- ACS Chemical Biology, Vol. 10, Issue 9
Practically Useful: What the R osetta Protein Modeling Suite Can Do for You
journal, April 2010
- Kaufmann, Kristian W.; Lemmon, Gordon H.; DeLuca, Samuel L.
- Biochemistry, Vol. 49, Issue 14
A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein
journal, February 2001
- Nakai, Junichi; Ohkura, Masamichi; Imoto, Keiji
- Nature Biotechnology, Vol. 19, Issue 2
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
Identification of Sites Within a Monomeric Red Fluorescent Protein that Tolerate Peptide Insertion and Testing of Corresponding Circular Permutations
journal, October 2007
- Li, Yankun; Sierra, Aillette M.; Ai, Hui-wang
- Photochemistry and Photobiology, Vol. 0, Issue 0
Zinc binding to a regulatory zinc-sensing domain monitored in vivo by using FRET
journal, May 2006
- Qiao, W.; Mooney, M.; Bird, A. J.
- Proceedings of the National Academy of Sciences, Vol. 103, Issue 23
An Expanded Palette of Genetically Encoded Ca2+ Indicators
journal, September 2011
- Zhao, Y.; Araki, S.; Wu, J.
- Science, Vol. 333, Issue 6051
Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging
journal, October 2012
- Akerboom, J.; Chen, T. -W.; Wardill, T. J.
- Journal of Neuroscience, Vol. 32, Issue 40
Solution Structure of a Zap1 Zinc-responsive Domain Provides Insights into Metalloregulatory Transcriptional Repression in Saccharomyces cerevisiae
journal, April 2006
- Wang, Zhonghua; Feng, Linda S.; Matskevich, Viktor
- Journal of Molecular Biology, Vol. 357, Issue 4
Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish
journal, March 2011
- Muto, A.; Ohkura, M.; Kotani, T.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 13
Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells
journal, May 2002
- Zacharias, D. A.
- Science, Vol. 296, Issue 5569
Engineering and characterization of a superfolder green fluorescent protein
journal, December 2005
- Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy
- Nature Biotechnology, Vol. 24, Issue 1, p. 79-88
Undetectable Intracellular Free Copper: The Requirement of a Copper Chaperone for Superoxide Dismutase
journal, April 1999
- Rae, T. D.
- Science, Vol. 284, Issue 5415
Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters
journal, September 2006
- Allen, Michael D.; Zhang, Jin
- Biochemical and Biophysical Research Communications, Vol. 348, Issue 2
A genetically targeted optical sensor to monitor calcium signals in astrocyte processes
journal, May 2010
- Shigetomi, Eiji; Kracun, Sebastian; Sofroniew, Michael V.
- Nature Neuroscience, Vol. 13, Issue 6
Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics
journal, January 2013
- Akerboom, Jasper; Carreras Calderón, Nicole; Tian, Lin
- Frontiers in Molecular Neuroscience, Vol. 6
Standards for photoluminescence quantum yield measurements in solution (IUPAC Technical Report)
journal, August 2011
- Brouwer, Albert M.
- Pure and Applied Chemistry, Vol. 83, Issue 12
Thermal lens measurement of absolute quantum yields using quenched fluorescent samples as references
journal, March 1989
- Shen, Jun; Snook, Richard D.
- Chemical Physics Letters, Vol. 155, Issue 6
Tris(2-pyridylmethyl)amine (TPA) as a membrane-permeable chelator for interception of biological mobile zinc
journal, January 2013
- Huang, Zhen; Zhang, Xiao-an; Bosch, Miquel
- Metallomics, Vol. 5, Issue 6
Works referencing / citing this record:
Genetically encoded single circularly permuted fluorescent protein-based intensity indicators
journal, January 2020
- Liu, Wenfeng; Deng, Mengying; Yang, Chengming
- Journal of Physics D: Applied Physics, Vol. 53, Issue 11
MerR‐fluorescent protein chimera biosensor for fast and sensitive detection of Hg 2+ in drinking water
journal, June 2019
- Özyurt, Canan; Üstükarcı, Handan; Evran, Serap
- Biotechnology and Applied Biochemistry, Vol. 66, Issue 5
Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification
journal, August 2019
- Kostyuk, Alexander I.; Demidovich, Aleksandra D.; Kotova, Daria A.
- International Journal of Molecular Sciences, Vol. 20, Issue 17
Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn2+ signals
journal, October 2019
- Minckley, Taylor F.; Zhang, Chen; Fudge, Dylan H.
- Nature Communications, Vol. 10, Issue 1
Spying on protein interactions in living cells with reconstituted scarlet light
journal, January 2018
- Wang, Sheng; Ding, Miao; Xue, Boxin
- The Analyst, Vol. 143, Issue 21
Sensors for measuring subcellular zinc pools
journal, January 2018
- Chabosseau, Pauline; Woodier, Jason; Cheung, Rebecca
- Metallomics, Vol. 10, Issue 2
Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn2+ signals
journal, October 2019
- Minckley, Taylor F.; Zhang, Chen; Fudge, Dylan H.
- Nature Communications, Vol. 10, Issue 1
Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification
journal, August 2019
- Kostyuk, Alexander I.; Demidovich, Aleksandra D.; Kotova, Daria A.
- International Journal of Molecular Sciences, Vol. 20, Issue 17