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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...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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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. |
format | Online Article Text |
id | pubmed-10454184 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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