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Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding
ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential prot...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226517/ https://www.ncbi.nlm.nih.gov/pubmed/32340357 http://dx.doi.org/10.3390/biom10040656 |
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author | Moore, Sydney Pickens, Alyssa Rodriguez, Jessica L. Marsee, Justin D. Miller, Justin M. |
author_facet | Moore, Sydney Pickens, Alyssa Rodriguez, Jessica L. Marsee, Justin D. Miller, Justin M. |
author_sort | Moore, Sydney |
collection | PubMed |
description | ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential protein substrates. Thus, these molecular machines must demonstrate greater stability than their substrates in order to ensure continued function in essential quality control networks. We report here a role for ATP in the stabilization of the inner membrane YME1L protease. Qualitative fluorescence data derived from protein unfolding experiments with urea reveal non-standard protein unfolding behavior that is dependent on [ATP]. Using multiple fluorophore systems, stopped-flow fluorescence experiments demonstrate a depletion of the native YME1L ensemble by urea-dependent unfolding and formation of a non-native conformation. Additional stopped-flow fluorescence experiments based on nucleotide binding and unfoldase activities predict that unfolding yields significant loss of active YME1L hexamers from the starting ensemble. Taken together, these data clearly define the stress limits of an important mitochondrial protease. |
format | Online Article Text |
id | pubmed-7226517 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-72265172020-05-18 Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding Moore, Sydney Pickens, Alyssa Rodriguez, Jessica L. Marsee, Justin D. Miller, Justin M. Biomolecules Article ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential protein substrates. Thus, these molecular machines must demonstrate greater stability than their substrates in order to ensure continued function in essential quality control networks. We report here a role for ATP in the stabilization of the inner membrane YME1L protease. Qualitative fluorescence data derived from protein unfolding experiments with urea reveal non-standard protein unfolding behavior that is dependent on [ATP]. Using multiple fluorophore systems, stopped-flow fluorescence experiments demonstrate a depletion of the native YME1L ensemble by urea-dependent unfolding and formation of a non-native conformation. Additional stopped-flow fluorescence experiments based on nucleotide binding and unfoldase activities predict that unfolding yields significant loss of active YME1L hexamers from the starting ensemble. Taken together, these data clearly define the stress limits of an important mitochondrial protease. MDPI 2020-04-23 /pmc/articles/PMC7226517/ /pubmed/32340357 http://dx.doi.org/10.3390/biom10040656 Text en © 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Moore, Sydney Pickens, Alyssa Rodriguez, Jessica L. Marsee, Justin D. Miller, Justin M. Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding |
title | Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding |
title_full | Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding |
title_fullStr | Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding |
title_full_unstemmed | Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding |
title_short | Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding |
title_sort | fluorescence methods applied to the description of urea-dependent yme1l protease unfolding |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226517/ https://www.ncbi.nlm.nih.gov/pubmed/32340357 http://dx.doi.org/10.3390/biom10040656 |
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