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

Bischofberger, Mirko; Iacovache, Mircea Ioan; Boss, Daniel; Naef, Felix; van der Goot, F Gisou; Molina, Nacho (2016). Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling. Biophysical journal, 110(7), pp. 1574-1581. Biophysical Society 10.1016/j.bpj.2016.02.035

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Anatomy

UniBE Contributor:

Iacovache, Mircea Ioan

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

0006-3495

Publisher:

Biophysical Society

Language:

English

Submitter:

Benoît Zuber

Date Deposited:

09 Jun 2016 14:18

Last Modified:

10 Jun 2016 11:28

Publisher DOI:

10.1016/j.bpj.2016.02.035

PubMed ID:

27074682

BORIS DOI:

10.7892/boris.81352

URI:

https://boris.unibe.ch/id/eprint/81352

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