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A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions

We developed a new method to analyze protein–protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein–protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisome...

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Autores principales: Murakami, Agnieszka M., Nagatomo, Katsuhiro, Miyoshi, Ichro, Itagaki, Shirou, Niwa, Yasutaka, Murakami, Manabu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10460381/
https://www.ncbi.nlm.nih.gov/pubmed/37634019
http://dx.doi.org/10.1038/s41598-023-41168-4
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author Murakami, Agnieszka M.
Nagatomo, Katsuhiro
Miyoshi, Ichro
Itagaki, Shirou
Niwa, Yasutaka
Murakami, Manabu
author_facet Murakami, Agnieszka M.
Nagatomo, Katsuhiro
Miyoshi, Ichro
Itagaki, Shirou
Niwa, Yasutaka
Murakami, Manabu
author_sort Murakami, Agnieszka M.
collection PubMed
description We developed a new method to analyze protein–protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein–protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein–protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 subunit (Ca(V)1.2) for the voltage-dependent calcium channel β2 subunit. Prokaryotic expression screening of the β2 subunit using an epitope library of Ca(V)1.2 resulted in two overlapping clones of the C-terminal sequence of Ca(V)1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of Ca(V)1.2 and β2.
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spelling pubmed-104603812023-08-28 A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions Murakami, Agnieszka M. Nagatomo, Katsuhiro Miyoshi, Ichro Itagaki, Shirou Niwa, Yasutaka Murakami, Manabu Sci Rep Article We developed a new method to analyze protein–protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein–protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein–protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 subunit (Ca(V)1.2) for the voltage-dependent calcium channel β2 subunit. Prokaryotic expression screening of the β2 subunit using an epitope library of Ca(V)1.2 resulted in two overlapping clones of the C-terminal sequence of Ca(V)1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of Ca(V)1.2 and β2. Nature Publishing Group UK 2023-08-26 /pmc/articles/PMC10460381/ /pubmed/37634019 http://dx.doi.org/10.1038/s41598-023-41168-4 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Murakami, Agnieszka M.
Nagatomo, Katsuhiro
Miyoshi, Ichro
Itagaki, Shirou
Niwa, Yasutaka
Murakami, Manabu
A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions
title A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions
title_full A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions
title_fullStr A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions
title_full_unstemmed A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions
title_short A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 subunit discovered using a new analysis method for protein–protein interactions
title_sort novel binding site between the voltage-dependent calcium channel ca(v)1.2 subunit and ca(v)β2 subunit discovered using a new analysis method for protein–protein interactions
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10460381/
https://www.ncbi.nlm.nih.gov/pubmed/37634019
http://dx.doi.org/10.1038/s41598-023-41168-4
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