Disassembly of IM30 higher-order oligomeric structures enables phase separation
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Abstract
Cyanobacteria are the most ancient oxygenic photosynthetic organisms that can convert the energy from the sun into biochemical energy. The protein complexes that are involved in the light-driven reactions are embedded in a specialized internal membrane system, the thylakoid membrane (TM). For the most part, biogenesis and maintenance of the TM system remains mysterious, yet clearly involves the inner membrane associated protein of 30 kDa (IM30). IM30 belongs to a superfamily of proteins, all members of which form large homo oligomeric structures and are involved in membrane remodeling.
First, the structural changes upon disassembly of oligomeric IM30 structures at increasing urea concentrations were investigated. To follow the unfolding process on different levels, we applied static light scattering, tryptophane fluorescence spectroscopy, circular dichroism spectroscopy together with molecular dynamics simulations. By developing and applying a 3-state model for IM30 oligomer disassembly we could show that IM30 remains structured in its conserved coiled-coil region after barrel disassembly while the rest of the protein already unfolds.
In the next section of this thesis, we show that disassembly of higher ordered oligomers enables IM30 to form biomolecular condensates in vitro. Furthermore, IM30 variant that is deficient in the formation of prototypical higher order oligomers still accumulated into so called punctae in living cells an. These punctae, which appear to be important for the in vivo function of IM30, likely represent IM30 condensates forming in vivo.
In the last section of this thesis, the interconnections between truncated IM30 variants and their ability to form condensates in vitro and punctae in vivo was investigated. We found that the structured coiled-coil region, but not an intrinsically disordered region, drives condensate formation of IM30, and the N-terminal coiled-coil-containing region in fact is also sufficient for puncta formation in vivo.