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CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones

The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of th...

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Autores principales: Klein, Florencia, Sardi, Florencia, Machado, Matías R., Ortega, Claudia, Comini, Marcelo A., Pantano, Sergio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991088/
https://www.ncbi.nlm.nih.gov/pubmed/33778003
http://dx.doi.org/10.3389/fmolb.2021.629773
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author Klein, Florencia
Sardi, Florencia
Machado, Matías R.
Ortega, Claudia
Comini, Marcelo A.
Pantano, Sergio
author_facet Klein, Florencia
Sardi, Florencia
Machado, Matías R.
Ortega, Claudia
Comini, Marcelo A.
Pantano, Sergio
author_sort Klein, Florencia
collection PubMed
description The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of the difficulties associated with the large size of the polypeptides involved, and their intrinsically huge conformational variability. Indeed, FRET sensors’ design still relies on vague structural notions, and trial and error combinations of linkers and protein modules. We recently designed a FRET sensor for the second messenger cAMP named CUTie (Cyclic nucleotide Universal Tag for imaging experiments), which granted sub-micrometer resolution in living cells. Here we apply a combination of sequence/structure analysis to produce a new-generation FRET sensor for the second messenger cGMP based on Protein kinase G I (PKGI), which we named CUTie2. Coarse-grained molecular dynamics simulations achieved an exhaustive sampling of the relevant spatio-temporal coordinates providing a quasi-quantitative prediction of the FRET efficiency, as confirmed by in vitro experiments. Moreover, biochemical characterization showed that the cGMP binding module maintains virtually the same affinity and selectivity for its ligand thant the full-length protein. The computational approach proposed here is easily generalizable to other allosteric protein modules, providing a cost effective-strategy for the custom design of FRET sensors.
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spelling pubmed-79910882021-03-26 CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones Klein, Florencia Sardi, Florencia Machado, Matías R. Ortega, Claudia Comini, Marcelo A. Pantano, Sergio Front Mol Biosci Molecular Biosciences The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of the difficulties associated with the large size of the polypeptides involved, and their intrinsically huge conformational variability. Indeed, FRET sensors’ design still relies on vague structural notions, and trial and error combinations of linkers and protein modules. We recently designed a FRET sensor for the second messenger cAMP named CUTie (Cyclic nucleotide Universal Tag for imaging experiments), which granted sub-micrometer resolution in living cells. Here we apply a combination of sequence/structure analysis to produce a new-generation FRET sensor for the second messenger cGMP based on Protein kinase G I (PKGI), which we named CUTie2. Coarse-grained molecular dynamics simulations achieved an exhaustive sampling of the relevant spatio-temporal coordinates providing a quasi-quantitative prediction of the FRET efficiency, as confirmed by in vitro experiments. Moreover, biochemical characterization showed that the cGMP binding module maintains virtually the same affinity and selectivity for its ligand thant the full-length protein. The computational approach proposed here is easily generalizable to other allosteric protein modules, providing a cost effective-strategy for the custom design of FRET sensors. Frontiers Media S.A. 2021-03-11 /pmc/articles/PMC7991088/ /pubmed/33778003 http://dx.doi.org/10.3389/fmolb.2021.629773 Text en Copyright © 2021 Klein, Sardi, Machado, Ortega, Comini and Pantano. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Klein, Florencia
Sardi, Florencia
Machado, Matías R.
Ortega, Claudia
Comini, Marcelo A.
Pantano, Sergio
CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones
title CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones
title_full CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones
title_fullStr CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones
title_full_unstemmed CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones
title_short CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones
title_sort cutie2: the attack of the cyclic nucleotide sensor clones
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991088/
https://www.ncbi.nlm.nih.gov/pubmed/33778003
http://dx.doi.org/10.3389/fmolb.2021.629773
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