Cargando…

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different c...

Descripción completa

Detalles Bibliográficos
Autores principales: Mizukami, Takuya, Abe, Yukiko, Maki, Kosuke
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526358/
https://www.ncbi.nlm.nih.gov/pubmed/26244984
http://dx.doi.org/10.1371/journal.pone.0134238
_version_ 1782384407738318848
author Mizukami, Takuya
Abe, Yukiko
Maki, Kosuke
author_facet Mizukami, Takuya
Abe, Yukiko
Maki, Kosuke
author_sort Mizukami, Takuya
collection PubMed
description In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.
format Online
Article
Text
id pubmed-4526358
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-45263582015-08-12 Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments Mizukami, Takuya Abe, Yukiko Maki, Kosuke PLoS One Research Article In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin. Public Library of Science 2015-08-05 /pmc/articles/PMC4526358/ /pubmed/26244984 http://dx.doi.org/10.1371/journal.pone.0134238 Text en © 2015 Mizukami et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Mizukami, Takuya
Abe, Yukiko
Maki, Kosuke
Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments
title Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments
title_full Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments
title_fullStr Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments
title_full_unstemmed Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments
title_short Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments
title_sort evidence for a shared mechanism in the formation of urea-induced kinetic and equilibrium intermediates of horse apomyoglobin from ultrarapid mixing experiments
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526358/
https://www.ncbi.nlm.nih.gov/pubmed/26244984
http://dx.doi.org/10.1371/journal.pone.0134238
work_keys_str_mv AT mizukamitakuya evidenceforasharedmechanismintheformationofureainducedkineticandequilibriumintermediatesofhorseapomyoglobinfromultrarapidmixingexperiments
AT abeyukiko evidenceforasharedmechanismintheformationofureainducedkineticandequilibriumintermediatesofhorseapomyoglobinfromultrarapidmixingexperiments
AT makikosuke evidenceforasharedmechanismintheformationofureainducedkineticandequilibriumintermediatesofhorseapomyoglobinfromultrarapidmixingexperiments