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

Abstract

Summary Protein 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.

Plastid 2-Cys peroxiredoxins are essential for embryogenesis in Arabidopsis

“…S2A), suggesting the presence of these enzymes in aggregates localized at defined locations in plastids of developing seeds. Moreover, the formation of 2-Cys Prx aggregates, hence the chaperone activity of the enzyme, requires the peroxidatic Cys residue at its active site [62,63]. The failure of the 2CPA-C P -S mutant version of 2-Cys Prx A, in which the peroxidatic Cys residue was replaced by Ser, to recover normal embryogenesis and seed development when expressed in the 2cpab mutant background (Fig.…”

Mitochondrial Peroxiredoxin-IIF (PRXIIF) Activity and Function during Seed Aging

“…PRXs exert signaling functions, e.g., by acting as cell cycle regulators [ 60 ] or by regulating metabolism [ 61 ]. These functions depend on certain molecular mechanisms such as (i) formation of intermolecular disulfide bonds with partner proteins, (ii) controlling the redox state of redox transmitters that, in turn, switch target proteins, (iii) functioning as chaperone proteins or (iv) interacting in a redox-state-dependent manner with proteins, as shown by interactome proteomics, e.g., for the chloroplast 2-CysPRX [ 5 , 59 ].…”

“…Other PRX isoforms provide defense against oxidative damage, and others are involved in signal transduction by controlling the H 2 O 2 concentration [ 4 ]. Thiol oxidation of certain PRXs leads to the formation of macromolecular multimers [ 5 ] that adopt molecular chaperone activity. The oxidized form of PRX cannot function as peroxide reductase [ 6 ].…”

A Photonic Resonator Interferometric Scattering Microscope for Label-free Detection of Nanometer-Scale Objects with Digital Precision in Point-of-Use Environments

“…The ability to detect and characterize biological nano-objects at digital resolution without extrinsic labels or labor-intensive assay preparation is the core advantage of interferometric scattering microscopy (iSCAT). Since its introduction nearly two decades ago [1], iSCAT has attracted significant interest in the field of bioimaging applications, including biomolecular and nanomaterial mass photometry [2][3][4], protein interaction and oligomerization characterization [5,6], three-dimensional particle tracking, and nanoparticle classification [7,8]. Imaging contrast in iSCAT relies on the fact that the detected signal is linearly dependent upon the volume of the scattering particle rather than the volume squared, as for pure scattering-based microscopies.…”