---
_id: '1611'
abstract:
- lang: eng
text: Biosensors for signaling molecules allow the study of physiological processes
by bringing together the fields of protein engineering, fluorescence imaging,
and cell biology. Construction of genetically encoded biosensors generally relies
on the availability of a binding "core" that is both specific and stable,
which can then be combined with fluorescent molecules to create a sensor. However,
binding proteins with the desired properties are often not available in nature
and substantial improvement to sensors can be required, particularly with regard
to their durability. Ancestral protein reconstruction is a powerful protein-engineering
tool able to generate highly stable and functional proteins. In this work, we
sought to establish the utility of ancestral protein reconstruction to biosensor
development, beginning with the construction of an l-arginine biosensor. l-arginine,
as the immediate precursor to nitric oxide, is an important molecule in many physiological
contexts including brain function. Using a combination of ancestral reconstruction
and circular permutation, we constructed a Förster resonance energy transfer (FRET)
biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity,
with a Kd of ∼14 μM and a maximal dynamic range of 35%. Importantly, cpFLIPR was
highly robust, enabling accurate l-arginine measurement at physiological temperatures.
We established that cpFLIPR is compatible with two-photon excitation fluorescence
microscopy and report l-arginine concentrations in brain tissue.
author:
- first_name: Jason
full_name: Whitfield, Jason
last_name: Whitfield
- first_name: William
full_name: Zhang, William
last_name: Zhang
- first_name: Michel
full_name: Herde, Michel
last_name: Herde
- first_name: Ben
full_name: Clifton, Ben
last_name: Clifton
- first_name: Johanna
full_name: Radziejewski, Johanna
last_name: Radziejewski
- first_name: Harald L
full_name: Janovjak, Harald L
id: 33BA6C30-F248-11E8-B48F-1D18A9856A87
last_name: Janovjak
orcid: 0000-0002-8023-9315
- first_name: Christian
full_name: Henneberger, Christian
last_name: Henneberger
- first_name: Colin
full_name: Jackson, Colin
last_name: Jackson
citation:
ama: Whitfield J, Zhang W, Herde M, et al. Construction of a robust and sensitive
arginine biosensor through ancestral protein reconstruction. Protein Science.
2015;24(9):1412-1422. doi:10.1002/pro.2721
apa: Whitfield, J., Zhang, W., Herde, M., Clifton, B., Radziejewski, J., Janovjak,
H. L., … Jackson, C. (2015). Construction of a robust and sensitive arginine biosensor
through ancestral protein reconstruction. Protein Science. Wiley. https://doi.org/10.1002/pro.2721
chicago: Whitfield, Jason, William Zhang, Michel Herde, Ben Clifton, Johanna Radziejewski,
Harald L Janovjak, Christian Henneberger, and Colin Jackson. “Construction of
a Robust and Sensitive Arginine Biosensor through Ancestral Protein Reconstruction.”
Protein Science. Wiley, 2015. https://doi.org/10.1002/pro.2721.
ieee: J. Whitfield et al., “Construction of a robust and sensitive arginine
biosensor through ancestral protein reconstruction,” Protein Science, vol.
24, no. 9. Wiley, pp. 1412–1422, 2015.
ista: Whitfield J, Zhang W, Herde M, Clifton B, Radziejewski J, Janovjak HL, Henneberger
C, Jackson C. 2015. Construction of a robust and sensitive arginine biosensor
through ancestral protein reconstruction. Protein Science. 24(9), 1412–1422.
mla: Whitfield, Jason, et al. “Construction of a Robust and Sensitive Arginine Biosensor
through Ancestral Protein Reconstruction.” Protein Science, vol. 24, no.
9, Wiley, 2015, pp. 1412–22, doi:10.1002/pro.2721.
short: J. Whitfield, W. Zhang, M. Herde, B. Clifton, J. Radziejewski, H.L. Janovjak,
C. Henneberger, C. Jackson, Protein Science 24 (2015) 1412–1422.
date_created: 2018-12-11T11:53:01Z
date_published: 2015-09-01T00:00:00Z
date_updated: 2021-01-12T06:52:00Z
day: '01'
department:
- _id: HaJa
doi: 10.1002/pro.2721
external_id:
pmid:
- '26061224'
intvolume: ' 24'
issue: '9'
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570536/
month: '09'
oa: 1
oa_version: Submitted Version
page: 1412 - 1422
pmid: 1
project:
- _id: 255BFFFA-B435-11E9-9278-68D0E5697425
grant_number: RGY0084/2012
name: In situ real-time imaging of neurotransmitter signaling using designer optical
sensors (HFSP Young Investigator)
publication: Protein Science
publication_status: published
publisher: Wiley
publist_id: '5555'
quality_controlled: '1'
scopus_import: 1
status: public
title: Construction of a robust and sensitive arginine biosensor through ancestral
protein reconstruction
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 24
year: '2015'
...