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This content will become publicly available on July 28, 2017

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

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 » to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.« less
Authors:
 [1] ;  [2] ;  [1] ;  [1] ;  [1]
  1. Univ. of Colorado, Boulder, CO (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
OSTI Identifier:
1330942
Report Number(s):
NREL/JA--2700-66894
Journal ID: ISSN 1554-8929
Grant/Contract Number:
AC36-08GO28308
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
Research Org:
NREL (National Renewable Energy Laboratory (NREL), Golden, CO (United States))
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES zinc; genetically encoded sensor; single fluorescent protein; Rosetta