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Real-time single-molecule observation of chaperone-assisted protein folding

The ability of heat shock protein 70 (Hsp70) molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes tow...

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Autores principales: Marzano, Nicholas R., Paudel, Bishnu P., van Oijen, Antoine M., Ecroyd, Heath
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9750156/
https://www.ncbi.nlm.nih.gov/pubmed/36516244
http://dx.doi.org/10.1126/sciadv.add0922
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author Marzano, Nicholas R.
Paudel, Bishnu P.
van Oijen, Antoine M.
Ecroyd, Heath
author_facet Marzano, Nicholas R.
Paudel, Bishnu P.
van Oijen, Antoine M.
Ecroyd, Heath
author_sort Marzano, Nicholas R.
collection PubMed
description The ability of heat shock protein 70 (Hsp70) molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes toward efficient protein folding. Here, we used total internal reflection fluorescence (TIRF) microscopy and single-molecule fluorescence resonance energy transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase proteins as they are folded by the bacterial Hsp70 system. We observed multiple cycles of chaperone binding and release to an individual client during refolding and determined that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism, whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.
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spelling pubmed-97501562022-12-21 Real-time single-molecule observation of chaperone-assisted protein folding Marzano, Nicholas R. Paudel, Bishnu P. van Oijen, Antoine M. Ecroyd, Heath Sci Adv Biomedicine and Life Sciences The ability of heat shock protein 70 (Hsp70) molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes toward efficient protein folding. Here, we used total internal reflection fluorescence (TIRF) microscopy and single-molecule fluorescence resonance energy transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase proteins as they are folded by the bacterial Hsp70 system. We observed multiple cycles of chaperone binding and release to an individual client during refolding and determined that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism, whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches. American Association for the Advancement of Science 2022-12-14 /pmc/articles/PMC9750156/ /pubmed/36516244 http://dx.doi.org/10.1126/sciadv.add0922 Text en Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (https://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
spellingShingle Biomedicine and Life Sciences
Marzano, Nicholas R.
Paudel, Bishnu P.
van Oijen, Antoine M.
Ecroyd, Heath
Real-time single-molecule observation of chaperone-assisted protein folding
title Real-time single-molecule observation of chaperone-assisted protein folding
title_full Real-time single-molecule observation of chaperone-assisted protein folding
title_fullStr Real-time single-molecule observation of chaperone-assisted protein folding
title_full_unstemmed Real-time single-molecule observation of chaperone-assisted protein folding
title_short Real-time single-molecule observation of chaperone-assisted protein folding
title_sort real-time single-molecule observation of chaperone-assisted protein folding
topic Biomedicine and Life Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9750156/
https://www.ncbi.nlm.nih.gov/pubmed/36516244
http://dx.doi.org/10.1126/sciadv.add0922
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