Development of an Optical Zn2+ Probe Based on a Single Fluorescent Protein
Publication Date
8-12-2016
Document Type
Article
Organizational Units
College of Natural Science and Mathematics, Biological Sciences
Keywords
Fluorescent probes, Intracellular labile Zn2+, Protein, Green Zinc Probe
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). 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.
Copyright Date
8-12-2016
Copyright Statement / License for Reuse
All Rights Reserved.
Rights Holder
American Chemical Society
Provenance
Received from author
Language
English (eng)
Extent
8 pgs
Publication Statement
Copyright is held by the American Chemical Society. User is responsible for all copyright compliance.
Publication Title
ACS Chemical Biology
Volume
11
Issue
10
First Page
2744
Last Page
2751
ISSN
1554-8937
Recommended Citation
Qin, Y., Sammond, D. W., Braselmann, E., Carpenter, M. C., & Palmer, A. E. (2016). Development of an optical Zn2 probe based on a single fluorescent protein. ACS Chemical Biology, 11(10), 2744–2751. DOI: 10.1021/acschembio.6b00442