Characterization of heterologously expressed fibril filaments, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma

Abstract

Fibril is a constitutive filament forming cytoskeletal protein of unidentified fold, exclusive to members of genus Spiroplasma. It is hypothesized to undergo conformational changes necessary to bring about Spiroplasma motility through changes in body helicity. However, in the absence of a cofactor such as nucleotide that binds to the protein and drives polymerization, the mechanism driving conformational changes in fibril remains unknown. Sodium dodecyl sulphate (SDS) solubilized the fibril filaments and facilitated fibril purification by affinity chromatography. An alternate protocol for obtaining enriched insoluble fibril filaments has been standardized using density gradient centrifugation method. Visualization of purified protein using electron microscopy demonstrated that it forms filament bundles. Probable domain boundaries of fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of both -helical and {beta}-sheet signatures in FT-IR measurements suggests that fibril filaments consist of assembly of folded globular domains, and not a {beta}-strand based aggregation similar to amyloid fibrils.