skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Development of an optical Zn 2+ probe based on a single fluorescent protein

Abstract

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn 2+. Here we present Green Zinc Probe (GZnP), a novel genetically encoded Zn 2+ 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 Zn 2+ 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 Zn 2+ (F max/F minmore » = 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 Zn 2+ 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:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1330942
Report Number(s):
NREL/JA-2700-66894
Journal ID: ISSN 1554-8929
Grant/Contract Number:
AC36-08GO28308
Resource Type:
Journal Article: 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. doi:10.1021/acschembio.6b00442.
Qin, Yan, Sammond, Deanne W., Braselmann, Esther, Carpenter, Margaret C., and Palmer, Amy E.. 2016. "Development of an optical Zn2+ probe based on a single fluorescent protein". United States. doi: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
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:
  • Individual photophysical parameters of the chromophore of a fluorescent protein mRFP1 and its two mutants (amino-acid substitution at position 66 - mRFP1/ Q66C and mRFP1/Q66S proteins) are determined. For this purpose, apart from conventional methods of fluorimetry and spectrophotometry, nonlinear laser fluorimetry is used. It is shown that the individual extinction coefficients of the chromophore of proteins correlate (correlation coefficient above 0.9) with the volume of the substituted amino-acid residue at position 66 (similar to the positions of the absorption, fluorescence excitation and emission maxima). (laser biology and medicine)
  • A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, {sup 1}H NMR, and {sup 13}C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies.more » Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials.« less
  • Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei ofmore » fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.« less
  • We have synthesized a red biarsenical fluorescent probe, AsCy3, with good photostability, low pH sensitivity, high absorbance and good quantum yield. It is directed specifically to a small tetracysteine peptide binding motif Cy3TAG (CysCysLysAlaGluAlaAlaCysCys) in the presence of other tetracysteine tags. This new probe provides a FRET partner to biarsenical dye FlAsH, making this discovery an important step toward a whole toolkit of colored probes directed to different small peptide motifs.
  • Small-molecule biarsenical multiuse affinity probes (MAPs) FlAsH and ReAsH,1,2 in conjunction with complementary protein tags, are important new tools for analyzing cellular function through live-cell imaging,3,4 targeted protein inactivation,5 and the measurement of protein dynamics and binding.6 In addition, MAPs serve as affinity reagents for isolating intact protein complexes for complementary structural measurements.7 These first-generation MAPs bind to a tetracoordinate arsenic group (TAG) binding motif (i.e., CCXXCC or FlAsHTAG) genetically engineered onto a protein of interest. They are superior to other targeted labeling strategies (such as the Halo-tag, the SNAP tag, and fluorescent proteins) in that the small peptide tagmore » does not disrupt protein protein interactions nor perturb the correct trafficking of tagged proteins.8,9 The conserved interatomic distance (*6 Å) between the two arsenic moieties in FlAsH and ReAsH complicates the selective labeling of multiple proteins with different reporters. To overcome these limitations, we have synthesized a new biarsenical MAP (i.e., AsCy3) based on Cy3, a member of the cyanine dye family, whose well-recognized brightness and photostability facilitate their utility in single-molecule measurements. The large interatomic distance between the two arsenics in AsCy3 (*14.5 Å) coupled with the identification of a complementary high-affinity binding sequence CCKAEAACC (Cy3TAG) permits the simultaneous application of both AsCy3 and FlAsH to selectively label their respective binding TAGs in different proteins. In addition, the fluorescence of FlAsH overlaps with the absorption of AsCy3, which can act as an acceptor of fluorescence resonance energy transfer (FRET) to allow ratiometric measurements of protein association.« less