Single molecule mass photometry reveals the dynamic oligomerization of human and plant peroxiredoxins

Michael Liebthal,Manish Singh Kushwah,Philipp Kukura,Karl-Josef Dietz

doi:10.1016/j.isci.2021.103258

Michael Liebthal, Manish Singh Kushwah + Show 2 more

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https://doi.org/10.1016/j.isci.2021.103258

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Journal: iScience	Publication Date: Oct 13, 2021
Citations: 14	License type: cc-by

Affiliation: Bielefeld University, University of Oxford

Abstract

SummaryProtein oligomerization is central to biological function and regulation, yet its experimental quantification and measurement of dynamic transitions in solution remain challenging. Here, we show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution. We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs). Comparison of the polydispersity of plant and human 2CPs as a function of concentration and redox state revealed features conserved among all 2CPs. In addition, we also find species-specific differences in oligomeric transitions, the occurrence of intermediates and the formation of high molecular weight complexes, which are associated with chaperone activity or act as a storage pool for more efficient dimers outlining the functional differentiation of human 2CPs. Our results point to a diversified functionality of oligomerization for 2CPs and illustrate the power of mass photometry for characterizing heterogeneous oligomeric protein distributions in near native conditions.

Highlights

Protein oligomerization – crucial for biological function and regulation of proteins – is driven by both homotypic and heterotypic protein interactions
We show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution
We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs)

Summary

Introduction

Protein oligomerization – crucial for biological function and regulation of proteins – is driven by both homotypic and heterotypic protein interactions. Oligomerization is a broadly used term referring to a wide range of processes, such as dimerization and tetramerization, ring-like structure formation, macroscopic filament formation, or numerous intermediates of a functional protein assembly. 2-Cysteine peroxiredoxins (2CPs) are the largest subgroup in the family of peroxiredoxins (PRX) and are a prototypical example of the range and functional importance of oligomerization. They fulfill diverse functions across the kingdoms of life and within the same cell, depending on their degree of oligomerization, which in turn is controlled by their redox state (Wood et al, 2002; Dietz, 2011; Poole and Nelson, 2016). Mainly considered as thiol peroxidases to scavenge reactive oxygen species (ROS) under regular and stress conditions, their role in various cellular mechanisms with redox-dependent processes ranges from cell differentiation, tumor suppression, signal transduction, thioredoxin (TRX) oxidation and chaperone-dependent protein homeostasis to participation in stress resistance and disease control (Neumann et al, 2003; Jang et al, 2004; Hall et al, 2009; Yan et al, 2009; Vaseghi et al, 2018)

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