---
_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: '958'
abstract:
- lang: eng
text: Biosensors that exploit Forster resonance energy transfer (FRET) can be used
to visualize biological and physiological processes and are capable of providing
detailed information in both spatial and temporal dimensions. In a FRET-based
biosensor, substrate binding is associated with a change in the relative positions
of two fluorophores, leading to a change in FRET efficiency that may be observed
in the fluorescence spectrum. As a result, their design requires a ligand-binding
protein that exhibits a conformational change upon binding. However, not all ligand-binding
proteins produce responsive sensors upon conjugation to fluorescent proteins or
dyes, and identifying the optimum locations for the fluorophores often involves
labor-intensive iterative design or high-throughput screening. Combining the genetic
fusion of a fluorescent protein to the ligand-binding protein with site-specific
covalent attachment of a fluorescent dye can allow fine control over the positions
of the two fluorophores, allowing the construction of very sensitive sensors.
This relies upon the accurate prediction of the locations of the two fluorophores
in bound and unbound states. In this chapter, we describe a method for computational
identification of dye-attachment sites that allows the use of cysteine modification
to attach synthetic dyes that can be paired with a fluorescent protein for the
purposes of creating FRET sensors.
alternative_title:
- Methods in Molecular Biology
author:
- first_name: Joshua
full_name: Mitchell, Joshua
last_name: Mitchell
- first_name: William
full_name: Zhang, William
last_name: Zhang
- 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: Megan
full_name: O'Mara, Megan
last_name: O'Mara
- first_name: Colin
full_name: Jackson, Colin
last_name: Jackson
citation:
ama: 'Mitchell J, Zhang W, Herde M, et al. Method for developing optical sensors
using a synthetic dye fluorescent protein FRET pair and computational modeling
and assessment. In: Stein V, ed. Synthetic Protein Switches. Vol 1596.
Synthetic Protein Switches. Springer; 2017:89-99. doi:10.1007/978-1-4939-6940-1_6'
apa: Mitchell, J., Zhang, W., Herde, M., Henneberger, C., Janovjak, H. L., O’Mara,
M., & Jackson, C. (2017). Method for developing optical sensors using a synthetic
dye fluorescent protein FRET pair and computational modeling and assessment. In
V. Stein (Ed.), Synthetic Protein Switches (Vol. 1596, pp. 89–99). Springer.
https://doi.org/10.1007/978-1-4939-6940-1_6
chicago: Mitchell, Joshua, William Zhang, Michel Herde, Christian Henneberger, Harald
L Janovjak, Megan O’Mara, and Colin Jackson. “Method for Developing Optical Sensors
Using a Synthetic Dye Fluorescent Protein FRET Pair and Computational Modeling
and Assessment.” In Synthetic Protein Switches, edited by Viktor Stein,
1596:89–99. Synthetic Protein Switches. Springer, 2017. https://doi.org/10.1007/978-1-4939-6940-1_6.
ieee: J. Mitchell et al., “Method for developing optical sensors using a
synthetic dye fluorescent protein FRET pair and computational modeling and assessment,”
in Synthetic Protein Switches, vol. 1596, V. Stein, Ed. Springer, 2017,
pp. 89–99.
ista: 'Mitchell J, Zhang W, Herde M, Henneberger C, Janovjak HL, O’Mara M, Jackson
C. 2017.Method for developing optical sensors using a synthetic dye fluorescent
protein FRET pair and computational modeling and assessment. In: Synthetic Protein
Switches. Methods in Molecular Biology, vol. 1596, 89–99.'
mla: Mitchell, Joshua, et al. “Method for Developing Optical Sensors Using a Synthetic
Dye Fluorescent Protein FRET Pair and Computational Modeling and Assessment.”
Synthetic Protein Switches, edited by Viktor Stein, vol. 1596, Springer,
2017, pp. 89–99, doi:10.1007/978-1-4939-6940-1_6.
short: J. Mitchell, W. Zhang, M. Herde, C. Henneberger, H.L. Janovjak, M. O’Mara,
C. Jackson, in:, V. Stein (Ed.), Synthetic Protein Switches, Springer, 2017, pp.
89–99.
date_created: 2018-12-11T11:49:24Z
date_published: 2017-05-15T00:00:00Z
date_updated: 2021-01-12T08:22:13Z
day: '15'
department:
- _id: HaJa
doi: 10.1007/978-1-4939-6940-1_6
editor:
- first_name: Viktor
full_name: Stein, Viktor
last_name: Stein
intvolume: ' 1596'
language:
- iso: eng
month: '05'
oa_version: None
page: 89 - 99
publication: Synthetic Protein Switches
publication_identifier:
issn:
- '10643745'
publication_status: published
publisher: Springer
publist_id: '6450'
quality_controlled: '1'
scopus_import: 1
series_title: Synthetic Protein Switches
status: public
title: Method for developing optical sensors using a synthetic dye fluorescent protein
FRET pair and computational modeling and assessment
type: book_chapter
user_id: 4435EBFC-F248-11E8-B48F-1D18A9856A87
volume: 1596
year: '2017'
...
---
_id: '1024'
abstract:
- lang: eng
text: The history of auxin and cytokinin biology including the initial discoveries
by father–son duo Charles Darwin and Francis Darwin (1880), and Gottlieb Haberlandt
(1919) is a beautiful demonstration of unceasing continuity of research. Novel
findings are integrated into existing hypotheses and models and deepen our understanding
of biological principles. At the same time new questions are triggered and hand
to hand with this new methodologies are developed to address these new challenges.
alternative_title:
- Methods in Molecular Biology
author:
- first_name: Andrej
full_name: Hurny, Andrej
id: 4DC4AF46-F248-11E8-B48F-1D18A9856A87
last_name: Hurny
orcid: 0000-0003-3638-1426
- first_name: Eva
full_name: Benková, Eva
id: 38F4F166-F248-11E8-B48F-1D18A9856A87
last_name: Benková
orcid: 0000-0002-8510-9739
citation:
ama: Hurny A, Benková E. Methodological advances in auxin and cytokinin biology.
Auxins and Cytokinins in Plant Biology. 2017;1569:1-29. doi:10.1007/978-1-4939-6831-2_1
apa: Hurny, A., & Benková, E. (2017). Methodological advances in auxin and cytokinin
biology. Auxins and Cytokinins in Plant Biology. Springer. https://doi.org/10.1007/978-1-4939-6831-2_1
chicago: Hurny, Andrej, and Eva Benková. “Methodological Advances in Auxin and Cytokinin
Biology.” Auxins and Cytokinins in Plant Biology. Springer, 2017. https://doi.org/10.1007/978-1-4939-6831-2_1.
ieee: A. Hurny and E. Benková, “Methodological advances in auxin and cytokinin biology,”
Auxins and Cytokinins in Plant Biology, vol. 1569. Springer, pp. 1–29,
2017.
ista: Hurny A, Benková E. 2017. Methodological advances in auxin and cytokinin biology.
Auxins and Cytokinins in Plant Biology. 1569, 1–29.
mla: Hurny, Andrej, and Eva Benková. “Methodological Advances in Auxin and Cytokinin
Biology.” Auxins and Cytokinins in Plant Biology, vol. 1569, Springer,
2017, pp. 1–29, doi:10.1007/978-1-4939-6831-2_1.
short: A. Hurny, E. Benková, Auxins and Cytokinins in Plant Biology 1569 (2017)
1–29.
date_created: 2018-12-11T11:49:45Z
date_published: 2017-03-17T00:00:00Z
date_updated: 2024-03-25T23:30:09Z
day: '17'
ddc:
- '575'
department:
- _id: EvBe
doi: 10.1007/978-1-4939-6831-2_1
file:
- access_level: open_access
content_type: application/pdf
creator: system
date_created: 2018-12-12T10:14:18Z
date_updated: 2019-10-15T07:47:05Z
file_id: '5068'
file_name: IST-2018-1019-v1+1_Hurny_MethodsMolBiol_2017.pdf
file_size: 840646
relation: main_file
file_date_updated: 2019-10-15T07:47:05Z
has_accepted_license: '1'
intvolume: ' 1569'
language:
- iso: eng
month: '03'
oa: 1
oa_version: Submitted Version
page: 1 - 29
project:
- _id: 2542D156-B435-11E9-9278-68D0E5697425
call_identifier: FWF
grant_number: I 1774-B16
name: Hormone cross-talk drives nutrient dependent plant development
publication: Auxins and Cytokinins in Plant Biology
publication_identifier:
issn:
- '10643745'
publication_status: published
publisher: Springer
publist_id: '6369'
pubrep_id: '1019'
quality_controlled: '1'
related_material:
record:
- id: '539'
relation: dissertation_contains
status: public
scopus_import: 1
status: public
title: Methodological advances in auxin and cytokinin biology
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 1569
year: '2017'
...
---
_id: '2245'
abstract:
- lang: eng
text: 'Exogenous application of biologically important molecules for plant growth
promotion and/or regulation is very common both in plant research and horticulture.
Plant hormones such as auxins and cytokinins are classes of compounds which are
often applied exogenously. Nevertheless, plants possess a well-established machinery
to regulate the active pool of exogenously applied compounds by converting them
to metabolites and conjugates. Consequently, it is often very useful to know the
in vivo status of applied compounds to connect them with some of the regulatory
events in plant developmental processes. The in vivo status of applied compounds
can be measured by incubating plants with radiolabeled compounds, followed by
extraction, purification, and HPLC metabolic profiling of plant extracts. Recently
we have used this method to characterize the intracellularly localized PIN protein,
PIN5. Here we explain the method in detail, with a focus on general application. '
alternative_title:
- Methods in Molecular Biology
author:
- first_name: Sibu
full_name: Simon, Sibu
id: 4542EF9A-F248-11E8-B48F-1D18A9856A87
last_name: Simon
orcid: 0000-0002-1998-6741
- first_name: Petr
full_name: Skůpa, Petr
last_name: Skůpa
- first_name: Petre
full_name: Dobrev, Petre
last_name: Dobrev
- first_name: Jan
full_name: Petrášek, Jan
last_name: Petrášek
- first_name: Eva
full_name: Zažímalová, Eva
last_name: Zažímalová
- first_name: Jirí
full_name: Friml, Jirí
id: 4159519E-F248-11E8-B48F-1D18A9856A87
last_name: Friml
orcid: 0000-0002-8302-7596
citation:
ama: 'Simon S, Skůpa P, Dobrev P, Petrášek J, Zažímalová E, Friml J. Analyzing the
in vivo status of exogenously applied auxins: A HPLC-based method to characterize
the intracellularly localized auxin transporters. In: Hicks G, Robert S, eds.
Plant Chemical Genomics. Vol 1056. Methods in Molecular Biology. Springer;
2014:255-264. doi:10.1007/978-1-62703-592-7_23'
apa: 'Simon, S., Skůpa, P., Dobrev, P., Petrášek, J., Zažímalová, E., & Friml,
J. (2014). Analyzing the in vivo status of exogenously applied auxins: A HPLC-based
method to characterize the intracellularly localized auxin transporters. In G.
Hicks & S. Robert (Eds.), Plant Chemical Genomics (Vol. 1056, pp. 255–264).
Springer. https://doi.org/10.1007/978-1-62703-592-7_23'
chicago: 'Simon, Sibu, Petr Skůpa, Petre Dobrev, Jan Petrášek, Eva Zažímalová, and
Jiří Friml. “Analyzing the in Vivo Status of Exogenously Applied Auxins: A HPLC-Based
Method to Characterize the Intracellularly Localized Auxin Transporters.” In Plant
Chemical Genomics, edited by Glenn Hicks and Stéphanie Robert, 1056:255–64.
Methods in Molecular Biology. Springer, 2014. https://doi.org/10.1007/978-1-62703-592-7_23.'
ieee: 'S. Simon, P. Skůpa, P. Dobrev, J. Petrášek, E. Zažímalová, and J. Friml,
“Analyzing the in vivo status of exogenously applied auxins: A HPLC-based method
to characterize the intracellularly localized auxin transporters,” in Plant
Chemical Genomics, vol. 1056, G. Hicks and S. Robert, Eds. Springer, 2014,
pp. 255–264.'
ista: 'Simon S, Skůpa P, Dobrev P, Petrášek J, Zažímalová E, Friml J. 2014.Analyzing
the in vivo status of exogenously applied auxins: A HPLC-based method to characterize
the intracellularly localized auxin transporters. In: Plant Chemical Genomics.
Methods in Molecular Biology, vol. 1056, 255–264.'
mla: 'Simon, Sibu, et al. “Analyzing the in Vivo Status of Exogenously Applied Auxins:
A HPLC-Based Method to Characterize the Intracellularly Localized Auxin Transporters.”
Plant Chemical Genomics, edited by Glenn Hicks and Stéphanie Robert, vol.
1056, Springer, 2014, pp. 255–64, doi:10.1007/978-1-62703-592-7_23.'
short: S. Simon, P. Skůpa, P. Dobrev, J. Petrášek, E. Zažímalová, J. Friml, in:,
G. Hicks, S. Robert (Eds.), Plant Chemical Genomics, Springer, 2014, pp. 255–264.
date_created: 2018-12-11T11:56:32Z
date_published: 2014-01-01T00:00:00Z
date_updated: 2021-01-12T06:56:15Z
day: '01'
department:
- _id: JiFr
doi: 10.1007/978-1-62703-592-7_23
editor:
- first_name: Glenn
full_name: Hicks, Glenn
last_name: Hicks
- first_name: Stéphanie
full_name: Robert, Stéphanie
last_name: Robert
intvolume: ' 1056'
language:
- iso: eng
month: '01'
oa_version: None
page: 255 - 264
publication: Plant Chemical Genomics
publication_identifier:
issn:
- '10643745'
publication_status: published
publisher: Springer
publist_id: '4704'
quality_controlled: '1'
scopus_import: 1
series_title: Methods in Molecular Biology
status: public
title: 'Analyzing the in vivo status of exogenously applied auxins: A HPLC-based method
to characterize the intracellularly localized auxin transporters'
type: book_chapter
user_id: 4435EBFC-F248-11E8-B48F-1D18A9856A87
volume: 1056
year: '2014'
...