Cargando…

Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling

Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and olig...

Descripción completa

Detalles Bibliográficos
Autores principales: Bischofberger, Mirko, Iacovache, Ioan, Boss, Daniel, Naef, Felix, van der Goot, F. Gisou, Molina, Nacho
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Biophysical Society 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833779/
https://www.ncbi.nlm.nih.gov/pubmed/27074682
http://dx.doi.org/10.1016/j.bpj.2016.02.035
_version_ 1782427379636895744
author Bischofberger, Mirko
Iacovache, Ioan
Boss, Daniel
Naef, Felix
van der Goot, F. Gisou
Molina, Nacho
author_facet Bischofberger, Mirko
Iacovache, Ioan
Boss, Daniel
Naef, Felix
van der Goot, F. Gisou
Molina, Nacho
author_sort Bischofberger, Mirko
collection PubMed
description Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.
format Online
Article
Text
id pubmed-4833779
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher The Biophysical Society
record_format MEDLINE/PubMed
spelling pubmed-48337792017-04-12 Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling Bischofberger, Mirko Iacovache, Ioan Boss, Daniel Naef, Felix van der Goot, F. Gisou Molina, Nacho Biophys J Membranes Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways. The Biophysical Society 2016-04-12 2016-04-12 /pmc/articles/PMC4833779/ /pubmed/27074682 http://dx.doi.org/10.1016/j.bpj.2016.02.035 Text en © 2016 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Membranes
Bischofberger, Mirko
Iacovache, Ioan
Boss, Daniel
Naef, Felix
van der Goot, F. Gisou
Molina, Nacho
Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling
title Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling
title_full Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling
title_fullStr Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling
title_full_unstemmed Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling
title_short Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling
title_sort revealing assembly of a pore-forming complex using single-cell kinetic analysis and modeling
topic Membranes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833779/
https://www.ncbi.nlm.nih.gov/pubmed/27074682
http://dx.doi.org/10.1016/j.bpj.2016.02.035
work_keys_str_mv AT bischofbergermirko revealingassemblyofaporeformingcomplexusingsinglecellkineticanalysisandmodeling
AT iacovacheioan revealingassemblyofaporeformingcomplexusingsinglecellkineticanalysisandmodeling
AT bossdaniel revealingassemblyofaporeformingcomplexusingsinglecellkineticanalysisandmodeling
AT naeffelix revealingassemblyofaporeformingcomplexusingsinglecellkineticanalysisandmodeling
AT vandergootfgisou revealingassemblyofaporeformingcomplexusingsinglecellkineticanalysisandmodeling
AT molinanacho revealingassemblyofaporeformingcomplexusingsinglecellkineticanalysisandmodeling