Development of an Optical Zn2+ Probe Based on a Single Fluorescent Protein

Deanne Sammond, Yan Qin, Esther Braselmann, Margaret Carpenter, Amy Palmer

Research output: Contribution to journalArticlepeer-review

34 Scopus Citations

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.
Original languageAmerican English
Pages (from-to)2744-2751
Number of pages8
JournalACS Chemical Biology
Volume11
Issue number10
DOIs
StatePublished - 2016

NREL Publication Number

  • NREL/JA-2700-66894

Keywords

  • genetically encoded sensor
  • Rosetta
  • single fluorescent protein
  • zinc

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