Development of an optical Zn2+ probe based on a single fluorescent protein

Qin, Yan; Sammond, Deanne W.; Braselmann, Esther; Carpenter, Margaret C.; Palmer, Amy E.

doi:10.1021/acschembio.6b00442

Title: Development of an optical Zn²⁺ probe based on a single fluorescent protein

Journal Article · Thu Jul 28 00:00:00 EDT 2016 · ACS Chemical Biology

DOI:https://doi.org/10.1021/acschembio.6b00442· OSTI ID:1330942

Qin, Yan ^[1]; Sammond, Deanne W. ^[2]; Braselmann, Esther ^[1]; Carpenter, Margaret C. ^[1]; Palmer, Amy E. ^[1]

Univ. of Colorado, Boulder, CO (United States)
National Renewable Energy Lab. (NREL), Golden, CO (United States)

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 (F_max/F_min = 2.6, K_d = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn²⁺ (F_max/F_min = 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.

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Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1330942

Report Number(s):: NREL/JA-2700-66894

Journal Information:: ACS Chemical Biology, Vol. 11, Issue 10; ISSN 1554-8929

Publisher:: American Chemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 27 works

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Genetically encoded single circularly permuted fluorescent protein-based intensity indicators Liu, Wenfeng; Deng, Mengying; Yang, Chengming Journal of Physics D: Applied Physics, Vol. 53, Issue 11 https://doi.org/10.1088/1361-6463/ab5dd8	journal	January 2020
MerR‐fluorescent protein chimera biosensor for fast and sensitive detection of Hg ²⁺ in drinking water Özyurt, Canan; Üstükarcı, Handan; Evran, Serap Biotechnology and Applied Biochemistry, Vol. 66, Issue 5 https://doi.org/10.1002/bab.1805	journal	June 2019
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Spying on protein interactions in living cells with reconstituted scarlet light Wang, Sheng; Ding, Miao; Xue, Boxin The Analyst, Vol. 143, Issue 21 https://doi.org/10.1039/c8an01223g	journal	January 2018
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Title: Development of an optical Zn²⁺ probe based on a single fluorescent protein