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Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosens...

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Autores principales: Yagi-Utsumi, Maho, Miura, Haruko, Ganser, Christian, Watanabe, Hiroki, Hiranyakorn, Methanee, Satoh, Tadashi, Uchihashi, Takayuki, Kato, Koichi, Okazaki, Kei-ichi, Aoki, Kazuhiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10454184/
https://www.ncbi.nlm.nih.gov/pubmed/37629048
http://dx.doi.org/10.3390/ijms241612865
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author Yagi-Utsumi, Maho
Miura, Haruko
Ganser, Christian
Watanabe, Hiroki
Hiranyakorn, Methanee
Satoh, Tadashi
Uchihashi, Takayuki
Kato, Koichi
Okazaki, Kei-ichi
Aoki, Kazuhiro
author_facet Yagi-Utsumi, Maho
Miura, Haruko
Ganser, Christian
Watanabe, Hiroki
Hiranyakorn, Methanee
Satoh, Tadashi
Uchihashi, Takayuki
Kato, Koichi
Okazaki, Kei-ichi
Aoki, Kazuhiro
author_sort Yagi-Utsumi, Maho
collection PubMed
description Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b’ and a’ domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b’ and a’ domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.
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spelling pubmed-104541842023-08-26 Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status Yagi-Utsumi, Maho Miura, Haruko Ganser, Christian Watanabe, Hiroki Hiranyakorn, Methanee Satoh, Tadashi Uchihashi, Takayuki Kato, Koichi Okazaki, Kei-ichi Aoki, Kazuhiro Int J Mol Sci Article Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b’ and a’ domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b’ and a’ domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond. MDPI 2023-08-16 /pmc/articles/PMC10454184/ /pubmed/37629048 http://dx.doi.org/10.3390/ijms241612865 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yagi-Utsumi, Maho
Miura, Haruko
Ganser, Christian
Watanabe, Hiroki
Hiranyakorn, Methanee
Satoh, Tadashi
Uchihashi, Takayuki
Kato, Koichi
Okazaki, Kei-ichi
Aoki, Kazuhiro
Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status
title Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status
title_full Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status
title_fullStr Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status
title_full_unstemmed Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status
title_short Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status
title_sort molecular design of fret probes based on domain rearrangement of protein disulfide isomerase for monitoring intracellular redox status
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10454184/
https://www.ncbi.nlm.nih.gov/pubmed/37629048
http://dx.doi.org/10.3390/ijms241612865
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