---
_id: '137'
abstract:
- lang: eng
text: Fluorescent sensors are an essential part of the experimental toolbox of the
life sciences, where they are used ubiquitously to visualize intra- and extracellular
signaling. In the brain, optical neurotransmitter sensors can shed light on temporal
and spatial aspects of signal transmission by directly observing, for instance,
neurotransmitter release and spread. Here we report the development and application
of the first optical sensor for the amino acid glycine, which is both an inhibitory
neurotransmitter and a co-agonist of the N-methyl-d-aspartate receptors (NMDARs)
involved in synaptic plasticity. Computational design of a glycine-specific binding
protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can
be used with single and two-photon excitation fluorescence microscopy. We took
advantage of this newly developed sensor to test predictions about the uneven
spatial distribution of glycine in extracellular space and to demonstrate that
extracellular glycine levels are controlled by plasticity-inducing stimuli.
article_processing_charge: No
article_type: original
author:
- first_name: William
full_name: Zhang, William
last_name: Zhang
- first_name: Michel
full_name: Herde, Michel
last_name: Herde
- first_name: Joshua
full_name: Mitchell, Joshua
last_name: Mitchell
- first_name: Jason
full_name: Whitfield, Jason
last_name: Whitfield
- first_name: Andreas
full_name: Wulff, Andreas
last_name: Wulff
- first_name: Vanessa
full_name: Vongsouthi, Vanessa
last_name: Vongsouthi
- first_name: Inmaculada
full_name: Sanchez Romero, Inmaculada
id: 3D9C5D30-F248-11E8-B48F-1D18A9856A87
last_name: Sanchez Romero
- first_name: Polina
full_name: Gulakova, Polina
last_name: Gulakova
- first_name: Daniel
full_name: Minge, Daniel
last_name: Minge
- first_name: Björn
full_name: Breithausen, Björn
last_name: Breithausen
- first_name: Susanne
full_name: Schoch, Susanne
last_name: Schoch
- 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: Colin
full_name: Jackson, Colin
last_name: Jackson
- first_name: Christian
full_name: Henneberger, Christian
last_name: Henneberger
citation:
ama: Zhang W, Herde M, Mitchell J, et al. Monitoring hippocampal glycine with the
computationally designed optical sensor GlyFS. Nature Chemical Biology.
2018;14(9):861-869. doi:10.1038/s41589-018-0108-2
apa: Zhang, W., Herde, M., Mitchell, J., Whitfield, J., Wulff, A., Vongsouthi, V.,
… Henneberger, C. (2018). Monitoring hippocampal glycine with the computationally
designed optical sensor GlyFS. Nature Chemical Biology. Nature Publishing
Group. https://doi.org/10.1038/s41589-018-0108-2
chicago: Zhang, William, Michel Herde, Joshua Mitchell, Jason Whitfield, Andreas
Wulff, Vanessa Vongsouthi, Inmaculada Sanchez-Romero, et al. “Monitoring Hippocampal
Glycine with the Computationally Designed Optical Sensor GlyFS.” Nature Chemical
Biology. Nature Publishing Group, 2018. https://doi.org/10.1038/s41589-018-0108-2.
ieee: W. Zhang et al., “Monitoring hippocampal glycine with the computationally
designed optical sensor GlyFS,” Nature Chemical Biology, vol. 14, no. 9.
Nature Publishing Group, pp. 861–869, 2018.
ista: Zhang W, Herde M, Mitchell J, Whitfield J, Wulff A, Vongsouthi V, Sanchez-Romero
I, Gulakova P, Minge D, Breithausen B, Schoch S, Janovjak HL, Jackson C, Henneberger
C. 2018. Monitoring hippocampal glycine with the computationally designed optical
sensor GlyFS. Nature Chemical Biology. 14(9), 861–869.
mla: Zhang, William, et al. “Monitoring Hippocampal Glycine with the Computationally
Designed Optical Sensor GlyFS.” Nature Chemical Biology, vol. 14, no. 9,
Nature Publishing Group, 2018, pp. 861–69, doi:10.1038/s41589-018-0108-2.
short: W. Zhang, M. Herde, J. Mitchell, J. Whitfield, A. Wulff, V. Vongsouthi, I.
Sanchez-Romero, P. Gulakova, D. Minge, B. Breithausen, S. Schoch, H.L. Janovjak,
C. Jackson, C. Henneberger, Nature Chemical Biology 14 (2018) 861–869.
date_created: 2018-12-11T11:44:49Z
date_published: 2018-07-30T00:00:00Z
date_updated: 2023-09-13T08:58:05Z
day: '30'
department:
- _id: HaJa
doi: 10.1038/s41589-018-0108-2
external_id:
isi:
- '000442174500013'
pmid:
- '30061718 '
intvolume: ' 14'
isi: 1
issue: '9'
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://www.ncbi.nlm.nih.gov/pubmed/30061718
month: '07'
oa: 1
oa_version: Submitted Version
page: 861 - 869
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: Nature Chemical Biology
publication_status: published
publisher: Nature Publishing Group
publist_id: '7786'
quality_controlled: '1'
scopus_import: '1'
status: public
title: Monitoring hippocampal glycine with the computationally designed optical sensor
GlyFS
type: journal_article
user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1
volume: 14
year: '2018'
...
---
_id: '957'
abstract:
- lang: eng
text: Small molecule biosensors based on Forster resonance energy transfer (FRET)
enable small molecule signaling to be monitored with high spatial and temporal
resolution in complex cellular environments. FRET sensors can be constructed by
fusing a pair of fluorescent proteins to a suitable recognition domain, such as
a member of the solute-binding protein (SBP) superfamily. However, naturally occurring
SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability,
which may preclude imaging under physiological conditions, or because the positions
of their N- and C-termini may be suboptimal for fusion of fluorescent proteins,
which may limit the dynamic range of the resulting sensors. Here, we show how
these problems can be overcome using ancestral protein reconstruction and circular
permutation. Ancestral protein reconstruction, used as a protein engineering strategy,
leverages phylogenetic information to improve the thermostability of proteins,
while circular permutation enables the termini of an SBP to be repositioned to
maximize the dynamic range of the resulting FRET sensor. We also provide a protocol
for cloning the engineered SBPs into FRET sensor constructs using Golden Gate
assembly and discuss considerations for in situ characterization of the FRET sensors.
alternative_title:
- Methods in Molecular Biology
author:
- first_name: Ben
full_name: Clifton, Ben
last_name: Clifton
- first_name: Jason
full_name: Whitfield, Jason
last_name: Whitfield
- first_name: Inmaculada
full_name: Sanchez Romero, Inmaculada
id: 3D9C5D30-F248-11E8-B48F-1D18A9856A87
last_name: Sanchez Romero
- first_name: Michel
full_name: Herde, Michel
last_name: Herde
- first_name: Christian
full_name: Henneberger, Christian
last_name: Henneberger
- 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: Colin
full_name: Jackson, Colin
last_name: Jackson
citation:
ama: 'Clifton B, Whitfield J, Sanchez-Romero I, et al. Ancestral protein reconstruction
and circular permutation for improving the stability and dynamic range of FRET
sensors. In: Stein V, ed. Synthetic Protein Switches. Vol 1596. Synthetic
Protein Switches. Springer; 2017:71-87. doi:10.1007/978-1-4939-6940-1_5'
apa: Clifton, B., Whitfield, J., Sanchez-Romero, I., Herde, M., Henneberger, C.,
Janovjak, H. L., & Jackson, C. (2017). Ancestral protein reconstruction and
circular permutation for improving the stability and dynamic range of FRET sensors.
In V. Stein (Ed.), Synthetic Protein Switches (Vol. 1596, pp. 71–87). Springer.
https://doi.org/10.1007/978-1-4939-6940-1_5
chicago: Clifton, Ben, Jason Whitfield, Inmaculada Sanchez-Romero, Michel Herde,
Christian Henneberger, Harald L Janovjak, and Colin Jackson. “Ancestral Protein
Reconstruction and Circular Permutation for Improving the Stability and Dynamic
Range of FRET Sensors.” In Synthetic Protein Switches, edited by Viktor
Stein, 1596:71–87. Synthetic Protein Switches. Springer, 2017. https://doi.org/10.1007/978-1-4939-6940-1_5.
ieee: B. Clifton et al., “Ancestral protein reconstruction and circular permutation
for improving the stability and dynamic range of FRET sensors,” in Synthetic
Protein Switches, vol. 1596, V. Stein, Ed. Springer, 2017, pp. 71–87.
ista: 'Clifton B, Whitfield J, Sanchez-Romero I, Herde M, Henneberger C, Janovjak
HL, Jackson C. 2017.Ancestral protein reconstruction and circular permutation
for improving the stability and dynamic range of FRET sensors. In: Synthetic Protein
Switches. Methods in Molecular Biology, vol. 1596, 71–87.'
mla: Clifton, Ben, et al. “Ancestral Protein Reconstruction and Circular Permutation
for Improving the Stability and Dynamic Range of FRET Sensors.” Synthetic Protein
Switches, edited by Viktor Stein, vol. 1596, Springer, 2017, pp. 71–87, doi:10.1007/978-1-4939-6940-1_5.
short: B. Clifton, J. Whitfield, I. Sanchez-Romero, M. Herde, C. Henneberger, H.L.
Janovjak, C. Jackson, in:, V. Stein (Ed.), Synthetic Protein Switches, Springer,
2017, pp. 71–87.
date_created: 2018-12-11T11:49:24Z
date_published: 2017-03-15T00:00:00Z
date_updated: 2021-01-12T08:22:13Z
day: '15'
department:
- _id: HaJa
doi: 10.1007/978-1-4939-6940-1_5
editor:
- first_name: Viktor
full_name: Stein, Viktor
last_name: Stein
intvolume: ' 1596'
language:
- iso: eng
month: '03'
oa_version: None
page: 71 - 87
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: Synthetic Protein Switches
publication_identifier:
issn:
- '10643745'
publication_status: published
publisher: Springer
publist_id: '6451'
quality_controlled: '1'
scopus_import: 1
series_title: Synthetic Protein Switches
status: public
title: Ancestral protein reconstruction and circular permutation for improving the
stability and dynamic range of FRET sensors
type: book_chapter
user_id: 4435EBFC-F248-11E8-B48F-1D18A9856A87
volume: 1596
year: '2017'
...
---
_id: '1026'
abstract:
- lang: eng
text: The optogenetic revolution enabled spatially-precise and temporally-precise
control over protein function, signaling pathway activation, and animal behavior
with tremendous success in the dissection of signaling networks and neural circuits.
Very recently, optogenetic methods have been paired with optical reporters in
novel drug screening platforms. In these all-optical platforms, light remotely
activated ion channels and kinases thereby obviating the use of electrophysiology
or reagents. Consequences were remarkable operational simplicity, throughput,
and cost-effectiveness that culminated in the identification of new drug candidates.
These blueprints for all-optical assays also revealed potential pitfalls and inspire
all-optical variants of other screens, such as those that aim at better understanding
dynamic drug action or orphan protein function.
acknowledgement: This work was supported by grants of the European Union Seventh Framework
Programme (CIG-303564), the Human Frontier Science Program (RGY0084_2012), and the
Austrian Science Fund FWF (W1232 MolecularDrugTargets).
article_processing_charge: No
article_type: original
author:
- first_name: Viviana
full_name: Agus, Viviana
last_name: Agus
- first_name: Harald L
full_name: Janovjak, Harald L
id: 33BA6C30-F248-11E8-B48F-1D18A9856A87
last_name: Janovjak
orcid: 0000-0002-8023-9315
citation:
ama: 'Agus V, Janovjak HL. Optogenetic methods in drug screening: Technologies and
applications. Current Opinion in Biotechnology. 2017;48:8-14. doi:10.1016/j.copbio.2017.02.006'
apa: 'Agus, V., & Janovjak, H. L. (2017). Optogenetic methods in drug screening:
Technologies and applications. Current Opinion in Biotechnology. Elsevier.
https://doi.org/10.1016/j.copbio.2017.02.006'
chicago: 'Agus, Viviana, and Harald L Janovjak. “Optogenetic Methods in Drug Screening:
Technologies and Applications.” Current Opinion in Biotechnology. Elsevier,
2017. https://doi.org/10.1016/j.copbio.2017.02.006.'
ieee: 'V. Agus and H. L. Janovjak, “Optogenetic methods in drug screening: Technologies
and applications,” Current Opinion in Biotechnology, vol. 48. Elsevier,
pp. 8–14, 2017.'
ista: 'Agus V, Janovjak HL. 2017. Optogenetic methods in drug screening: Technologies
and applications. Current Opinion in Biotechnology. 48, 8–14.'
mla: 'Agus, Viviana, and Harald L. Janovjak. “Optogenetic Methods in Drug Screening:
Technologies and Applications.” Current Opinion in Biotechnology, vol.
48, Elsevier, 2017, pp. 8–14, doi:10.1016/j.copbio.2017.02.006.'
short: V. Agus, H.L. Janovjak, Current Opinion in Biotechnology 48 (2017) 8–14.
date_created: 2018-12-11T11:49:45Z
date_published: 2017-12-01T00:00:00Z
date_updated: 2023-09-22T09:26:06Z
day: '01'
department:
- _id: HaJa
doi: 10.1016/j.copbio.2017.02.006
ec_funded: 1
external_id:
isi:
- '000418313200003'
intvolume: ' 48'
isi: 1
language:
- iso: eng
month: '12'
oa_version: None
page: 8 - 14
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)
- _id: 25548C20-B435-11E9-9278-68D0E5697425
call_identifier: FP7
grant_number: '303564'
name: Microbial Ion Channels for Synthetic Neurobiology
- _id: 255A6082-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: W1232-B24
name: Molecular Drug Targets
publication: Current Opinion in Biotechnology
publication_identifier:
issn:
- '09581669'
publication_status: published
publisher: Elsevier
publist_id: '6365'
quality_controlled: '1'
scopus_import: '1'
status: public
title: 'Optogenetic methods in drug screening: Technologies and applications'
type: journal_article
user_id: c635000d-4b10-11ee-a964-aac5a93f6ac1
volume: 48
year: '2017'
...
---
_id: '1440'
acknowledgement: The author thanks Banerjee et al. (2016) for providing coordinates
prior to public release and apologizes to colleagues whose work was not cited or
discussed due to the limited space available. The author is supported by grants
from EU FP7 (CIG-303564), HFSP (RGY0084_2012), and FWF (W1232).
author:
- first_name: Harald L
full_name: Janovjak, Harald L
id: 33BA6C30-F248-11E8-B48F-1D18A9856A87
last_name: Janovjak
orcid: 0000-0002-8023-9315
citation:
ama: 'Janovjak HL. Light at the end of the protein: Crystal structure of a C-terminal
light-sensing domain. Structure. 2016;24(2):213-215. doi:10.1016/j.str.2016.01.002'
apa: 'Janovjak, H. L. (2016). Light at the end of the protein: Crystal structure
of a C-terminal light-sensing domain. Structure. Cell Press. https://doi.org/10.1016/j.str.2016.01.002'
chicago: 'Janovjak, Harald L. “Light at the End of the Protein: Crystal Structure
of a C-Terminal Light-Sensing Domain.” Structure. Cell Press, 2016. https://doi.org/10.1016/j.str.2016.01.002.'
ieee: 'H. L. Janovjak, “Light at the end of the protein: Crystal structure of a
C-terminal light-sensing domain,” Structure, vol. 24, no. 2. Cell Press,
pp. 213–215, 2016.'
ista: 'Janovjak HL. 2016. Light at the end of the protein: Crystal structure of
a C-terminal light-sensing domain. Structure. 24(2), 213–215.'
mla: 'Janovjak, Harald L. “Light at the End of the Protein: Crystal Structure of
a C-Terminal Light-Sensing Domain.” Structure, vol. 24, no. 2, Cell Press,
2016, pp. 213–15, doi:10.1016/j.str.2016.01.002.'
short: H.L. Janovjak, Structure 24 (2016) 213–215.
date_created: 2018-12-11T11:52:02Z
date_published: 2016-02-02T00:00:00Z
date_updated: 2021-01-12T06:50:46Z
day: '02'
department:
- _id: HaJa
doi: 10.1016/j.str.2016.01.002
ec_funded: 1
intvolume: ' 24'
issue: '2'
language:
- iso: eng
month: '02'
oa_version: None
page: 213 - 215
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)
- _id: 25548C20-B435-11E9-9278-68D0E5697425
call_identifier: FP7
grant_number: '303564'
name: Microbial Ion Channels for Synthetic Neurobiology
- _id: 255A6082-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: W1232-B24
name: Molecular Drug Targets
publication: Structure
publication_status: published
publisher: Cell Press
publist_id: '5756'
quality_controlled: '1'
scopus_import: 1
status: public
title: 'Light at the end of the protein: Crystal structure of a C-terminal light-sensing
domain'
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 24
year: '2016'
...
---
_id: '1678'
abstract:
- lang: eng
text: High-throughput live-cell screens are intricate elements of systems biology
studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted
method that avoids the need for chemical activators and reporters, reduces the
number of operational steps and increases information content in a cell-based
small-molecule screen against human protein kinases, including an orphan receptor
tyrosine kinase. This blueprint for all-optical screening can be adapted to many
drug targets and cellular processes.
acknowledgement: 'This work was supported by grants from the European Union Seventh
Framework Programme (CIG-303564 to H.J. and ERC-StG-311166 to S.M.B.N.), the Human
Frontier Science Program (RGY0084_2012 to H.J.) and the Herzfelder Foundation (to
M.G.). A.I.-P. was supported by a Ramon Areces fellowship, and E.R. by the graduate
program MolecularDrugTargets (Austrian Science Fund (FWF): W 1232) and a FemTech
fellowship (3580812 Austrian Research Promotion Agency).'
author:
- first_name: Álvaro
full_name: Inglés Prieto, Álvaro
id: 2A9DB292-F248-11E8-B48F-1D18A9856A87
last_name: Inglés Prieto
orcid: 0000-0002-5409-8571
- first_name: Eva
full_name: Gschaider-Reichhart, Eva
id: 3FEE232A-F248-11E8-B48F-1D18A9856A87
last_name: Gschaider-Reichhart
orcid: 0000-0002-7218-7738
- first_name: Markus
full_name: Muellner, Markus
last_name: Muellner
- first_name: Matthias
full_name: Nowak, Matthias
id: 30845DAA-F248-11E8-B48F-1D18A9856A87
last_name: Nowak
- first_name: Sebastian
full_name: Nijman, Sebastian
last_name: Nijman
- first_name: Michael
full_name: Grusch, Michael
last_name: Grusch
- first_name: Harald L
full_name: Janovjak, Harald L
id: 33BA6C30-F248-11E8-B48F-1D18A9856A87
last_name: Janovjak
orcid: 0000-0002-8023-9315
citation:
ama: Inglés Prieto Á, Gschaider-Reichhart E, Muellner M, et al. Light-assisted small-molecule
screening against protein kinases. Nature Chemical Biology. 2015;11(12):952-954.
doi:10.1038/nchembio.1933
apa: Inglés Prieto, Á., Gschaider-Reichhart, E., Muellner, M., Nowak, M., Nijman,
S., Grusch, M., & Janovjak, H. L. (2015). Light-assisted small-molecule screening
against protein kinases. Nature Chemical Biology. Nature Publishing Group.
https://doi.org/10.1038/nchembio.1933
chicago: Inglés Prieto, Álvaro, Eva Gschaider-Reichhart, Markus Muellner, Matthias
Nowak, Sebastian Nijman, Michael Grusch, and Harald L Janovjak. “Light-Assisted
Small-Molecule Screening against Protein Kinases.” Nature Chemical Biology.
Nature Publishing Group, 2015. https://doi.org/10.1038/nchembio.1933.
ieee: Á. Inglés Prieto et al., “Light-assisted small-molecule screening against
protein kinases,” Nature Chemical Biology, vol. 11, no. 12. Nature Publishing
Group, pp. 952–954, 2015.
ista: Inglés Prieto Á, Gschaider-Reichhart E, Muellner M, Nowak M, Nijman S, Grusch
M, Janovjak HL. 2015. Light-assisted small-molecule screening against protein
kinases. Nature Chemical Biology. 11(12), 952–954.
mla: Inglés Prieto, Álvaro, et al. “Light-Assisted Small-Molecule Screening against
Protein Kinases.” Nature Chemical Biology, vol. 11, no. 12, Nature Publishing
Group, 2015, pp. 952–54, doi:10.1038/nchembio.1933.
short: Á. Inglés Prieto, E. Gschaider-Reichhart, M. Muellner, M. Nowak, S. Nijman,
M. Grusch, H.L. Janovjak, Nature Chemical Biology 11 (2015) 952–954.
date_created: 2018-12-11T11:53:25Z
date_published: 2015-10-12T00:00:00Z
date_updated: 2023-09-07T12:49:09Z
day: '12'
ddc:
- '571'
department:
- _id: HaJa
- _id: LifeSc
doi: 10.1038/nchembio.1933
ec_funded: 1
file:
- access_level: open_access
checksum: e9fb251dfcb7cd209b83f17867e61321
content_type: application/pdf
creator: system
date_created: 2018-12-12T10:10:51Z
date_updated: 2020-07-14T12:45:12Z
file_id: '4842'
file_name: IST-2017-837-v1+1_ingles-prieto.pdf
file_size: 1308364
relation: main_file
file_date_updated: 2020-07-14T12:45:12Z
has_accepted_license: '1'
intvolume: ' 11'
issue: '12'
language:
- iso: eng
month: '10'
oa: 1
oa_version: Submitted Version
page: 952 - 954
project:
- _id: 25548C20-B435-11E9-9278-68D0E5697425
call_identifier: FP7
grant_number: '303564'
name: Microbial Ion Channels for Synthetic Neurobiology
- _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)
- _id: 255A6082-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: W1232-B24
name: Molecular Drug Targets
publication: Nature Chemical Biology
publication_status: published
publisher: Nature Publishing Group
publist_id: '5471'
pubrep_id: '837'
quality_controlled: '1'
related_material:
record:
- id: '418'
relation: dissertation_contains
status: public
scopus_import: 1
status: public
title: Light-assisted small-molecule screening against protein kinases
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 11
year: '2015'
...
---
_id: '1867'
abstract:
- lang: eng
text: Cultured mammalian cells essential are model systems in basic biology research,
production platforms of proteins for medical use, and testbeds in synthetic biology.
Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine
dinucleotide (FAD), are critical for cellular redox reactions and sense light
in naturally occurring photoreceptors and optogenetic tools. Here, we quantified
flavin contents of commonly used mammalian cell lines. We first compared three
procedures for extraction of free and noncovalently protein-bound flavins and
verified extraction using fluorescence spectroscopy. For separation, two CE methods
with different BGEs were established, and detection was performed by LED-induced
fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin
(RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14
amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN
(0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents
agree with those previously extracted from mammalian tissues, yet reduced forms
of RF were detected that were not described previously. Quantification of flavins
in mammalian cell lines will allow a better understanding of cellular redox reactions
and optogenetic tools.
author:
- first_name: Jens
full_name: Hühner, Jens
last_name: Hühner
- first_name: Álvaro
full_name: Inglés Prieto, Álvaro
id: 2A9DB292-F248-11E8-B48F-1D18A9856A87
last_name: Inglés Prieto
orcid: 0000-0002-5409-8571
- first_name: Christian
full_name: Neusüß, Christian
last_name: Neusüß
- first_name: Michael
full_name: Lämmerhofer, Michael
last_name: Lämmerhofer
- first_name: Harald L
full_name: Janovjak, Harald L
id: 33BA6C30-F248-11E8-B48F-1D18A9856A87
last_name: Janovjak
orcid: 0000-0002-8023-9315
citation:
ama: Hühner J, Inglés Prieto Á, Neusüß C, Lämmerhofer M, Janovjak HL. Quantification
of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian
model cells by CE with LED-induced fluorescence detection. Electrophoresis.
2015;36(4):518-525. doi:10.1002/elps.201400451
apa: Hühner, J., Inglés Prieto, Á., Neusüß, C., Lämmerhofer, M., & Janovjak,
H. L. (2015). Quantification of riboflavin, flavin mononucleotide, and flavin
adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence
detection. Electrophoresis. Wiley. https://doi.org/10.1002/elps.201400451
chicago: Hühner, Jens, Álvaro Inglés Prieto, Christian Neusüß, Michael Lämmerhofer,
and Harald L Janovjak. “Quantification of Riboflavin, Flavin Mononucleotide, and
Flavin Adenine Dinucleotide in Mammalian Model Cells by CE with LED-Induced Fluorescence
Detection.” Electrophoresis. Wiley, 2015. https://doi.org/10.1002/elps.201400451.
ieee: J. Hühner, Á. Inglés Prieto, C. Neusüß, M. Lämmerhofer, and H. L. Janovjak,
“Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide
in mammalian model cells by CE with LED-induced fluorescence detection,” Electrophoresis,
vol. 36, no. 4. Wiley, pp. 518–525, 2015.
ista: Hühner J, Inglés Prieto Á, Neusüß C, Lämmerhofer M, Janovjak HL. 2015. Quantification
of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian
model cells by CE with LED-induced fluorescence detection. Electrophoresis. 36(4),
518–525.
mla: Hühner, Jens, et al. “Quantification of Riboflavin, Flavin Mononucleotide,
and Flavin Adenine Dinucleotide in Mammalian Model Cells by CE with LED-Induced
Fluorescence Detection.” Electrophoresis, vol. 36, no. 4, Wiley, 2015,
pp. 518–25, doi:10.1002/elps.201400451.
short: J. Hühner, Á. Inglés Prieto, C. Neusüß, M. Lämmerhofer, H.L. Janovjak, Electrophoresis
36 (2015) 518–525.
date_created: 2018-12-11T11:54:26Z
date_published: 2015-02-01T00:00:00Z
date_updated: 2021-01-12T06:53:43Z
day: '01'
department:
- _id: HaJa
doi: 10.1002/elps.201400451
ec_funded: 1
intvolume: ' 36'
issue: '4'
language:
- iso: eng
month: '02'
oa_version: None
page: 518 - 525
project:
- _id: 25548C20-B435-11E9-9278-68D0E5697425
call_identifier: FP7
grant_number: '303564'
name: Microbial Ion Channels for Synthetic Neurobiology
- _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: Electrophoresis
publication_status: published
publisher: Wiley
publist_id: '5230'
pubrep_id: '836'
quality_controlled: '1'
scopus_import: 1
status: public
title: Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide
in mammalian model cells by CE with LED-induced fluorescence detection
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 36
year: '2015'
...
---
_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'
...
---
_id: '2857'
abstract:
- lang: eng
text: In the vibrant field of optogenetics, optics and genetic targeting are combined
to commandeer cellular functions, such as the neuronal action potential, by optically
stimulating light-sensitive ion channels expressed in the cell membrane. One broadly
applicable manifestation of this approach are covalently attached photochromic
tethered ligands (PTLs) that allow activating ligand-gated ion channels with outstanding
spatial and temporal resolution. Here, we describe all steps towards the successful
development and application of PTL-gated ion channels in cell lines and primary
cells. The basis for these experiments forms a combination of molecular modeling,
genetic engineering, cell culture, and electrophysiology. The light-gated glutamate
receptor (LiGluR), which consists of the PTL-functionalized GluK2 receptor, serves
as a model.
alternative_title:
- MIMB
author:
- first_name: Stephanie
full_name: Szobota, Stephanie
last_name: Szobota
- first_name: Catherine
full_name: Mckenzie, Catherine
id: 3EEDE19A-F248-11E8-B48F-1D18A9856A87
last_name: Mckenzie
- first_name: Harald L
full_name: Janovjak, Harald L
id: 33BA6C30-F248-11E8-B48F-1D18A9856A87
last_name: Janovjak
orcid: 0000-0002-8023-9315
citation:
ama: Szobota S, Mckenzie C, Janovjak HL. Optical control of ligand-gated ion channels.
Methods in Molecular Biology. 2013;998:417-435. doi:10.1007/978-1-62703-351-0_32
apa: Szobota, S., Mckenzie, C., & Janovjak, H. L. (2013). Optical control of
ligand-gated ion channels. Methods in Molecular Biology. Springer. https://doi.org/10.1007/978-1-62703-351-0_32
chicago: Szobota, Stephanie, Catherine Mckenzie, and Harald L Janovjak. “Optical
Control of Ligand-Gated Ion Channels.” Methods in Molecular Biology. Springer,
2013. https://doi.org/10.1007/978-1-62703-351-0_32.
ieee: S. Szobota, C. Mckenzie, and H. L. Janovjak, “Optical control of ligand-gated
ion channels,” Methods in Molecular Biology, vol. 998. Springer, pp. 417–435,
2013.
ista: Szobota S, Mckenzie C, Janovjak HL. 2013. Optical control of ligand-gated
ion channels. Methods in Molecular Biology. 998, 417–435.
mla: Szobota, Stephanie, et al. “Optical Control of Ligand-Gated Ion Channels.”
Methods in Molecular Biology, vol. 998, Springer, 2013, pp. 417–35, doi:10.1007/978-1-62703-351-0_32.
short: S. Szobota, C. Mckenzie, H.L. Janovjak, Methods in Molecular Biology 998
(2013) 417–435.
date_created: 2018-12-11T11:59:57Z
date_published: 2013-02-22T00:00:00Z
date_updated: 2021-01-12T07:00:17Z
day: '22'
ddc:
- '570'
department:
- _id: HaJa
doi: 10.1007/978-1-62703-351-0_32
ec_funded: 1
file:
- access_level: open_access
checksum: 1701f0d989f27ddac471b19a894ec0d1
content_type: application/pdf
creator: system
date_created: 2018-12-12T10:12:34Z
date_updated: 2020-07-14T12:45:51Z
file_id: '4952'
file_name: IST-2017-834-v1+1_szobota.pdf
file_size: 336734
relation: main_file
file_date_updated: 2020-07-14T12:45:51Z
has_accepted_license: '1'
intvolume: ' 998'
language:
- iso: eng
month: '02'
oa: 1
oa_version: Submitted Version
page: 417 - 435
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)
- _id: 25548C20-B435-11E9-9278-68D0E5697425
call_identifier: FP7
grant_number: '303564'
name: Microbial Ion Channels for Synthetic Neurobiology
publication: Methods in Molecular Biology
publication_status: published
publisher: Springer
publist_id: '3932'
pubrep_id: '834'
quality_controlled: '1'
scopus_import: 1
status: public
title: Optical control of ligand-gated ion channels
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 998
year: '2013'
...