Insights into assembly of constitutive filament-forming proteins of novel fold through domain dissection and EGFP insertion-based screen: A case study ofSpiroplasmaFibril

Author:

Bapat Mrinmayee,Harne ShrikantORCID,Kashyap Rajnandani,Gayathri PananghatORCID

Abstract

AbstractSpiroplasmais a cell-wall less, helical bacterium possessing a cytoskeletal ribbon consisting Fibril and 5 MreB paralogs. In absence of any information regarding the filament interface of Fibril, a cytoskeleton protein of novel fold, multiple short constructs of Fibril were designed to identify minimal domains required for polymerization. Purification trials of these short constructs resulted in insoluble protein products. In a parallel approach, we incorporated EGFP at the loop regions aimed to hinder polymerization and obtain non-polymerising constructs. We were able to identify probable interfilament/intrafilament interface residues based on the fluorescence screen. We show that EGFP insertions at many of the positions are tolerated as Fibril filaments were readily observed for these constructs in transmission electron microscopy. Both the approaches suggest that Fibril requires N and C-terminal domains for its polymerization. Earlier studies have shown that FibrilWTinteracts with MreB5. Our Fibril-EGFP constructs and MreB5 showed interaction similar to the wild-type. Co-sedimentation assay and visualization of Fibril-EGFP proteins proves that the fluorescent constructs are folded and functional and possess structural and biochemical properties similar to the wild type protein.

Publisher

Cold Spring Harbor Laboratory

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