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Extending fluorescence anisotropy to large complexes using reversibly switchable proteins
The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We descri...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group US
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10110461/ https://www.ncbi.nlm.nih.gov/pubmed/36217028 http://dx.doi.org/10.1038/s41587-022-01489-7 |
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author | Volpato, Andrea Ollech, Dirk Alvelid, Jonatan Damenti, Martina Müller, Barbara York, Andrew G Ingaramo, Maria Testa, Ilaria |
author_facet | Volpato, Andrea Ollech, Dirk Alvelid, Jonatan Damenti, Martina Müller, Barbara York, Andrew G Ingaramo, Maria Testa, Ilaria |
author_sort | Volpato, Andrea |
collection | PubMed |
description | The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome. |
format | Online Article Text |
id | pubmed-10110461 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group US |
record_format | MEDLINE/PubMed |
spelling | pubmed-101104612023-04-19 Extending fluorescence anisotropy to large complexes using reversibly switchable proteins Volpato, Andrea Ollech, Dirk Alvelid, Jonatan Damenti, Martina Müller, Barbara York, Andrew G Ingaramo, Maria Testa, Ilaria Nat Biotechnol Article The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome. Nature Publishing Group US 2022-10-10 2023 /pmc/articles/PMC10110461/ /pubmed/36217028 http://dx.doi.org/10.1038/s41587-022-01489-7 Text en © The Author(s) 2022 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Volpato, Andrea Ollech, Dirk Alvelid, Jonatan Damenti, Martina Müller, Barbara York, Andrew G Ingaramo, Maria Testa, Ilaria Extending fluorescence anisotropy to large complexes using reversibly switchable proteins |
title | Extending fluorescence anisotropy to large complexes using reversibly switchable proteins |
title_full | Extending fluorescence anisotropy to large complexes using reversibly switchable proteins |
title_fullStr | Extending fluorescence anisotropy to large complexes using reversibly switchable proteins |
title_full_unstemmed | Extending fluorescence anisotropy to large complexes using reversibly switchable proteins |
title_short | Extending fluorescence anisotropy to large complexes using reversibly switchable proteins |
title_sort | extending fluorescence anisotropy to large complexes using reversibly switchable proteins |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10110461/ https://www.ncbi.nlm.nih.gov/pubmed/36217028 http://dx.doi.org/10.1038/s41587-022-01489-7 |
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