Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma
Summary: 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 cell helicity. However, the mechanism driving...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-10-01
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| Series: | iScience |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S258900422201327X |
| _version_ | 1798032803072311296 |
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| author | Shrikant Harne Pananghat Gayathri |
| author_facet | Shrikant Harne Pananghat Gayathri |
| author_sort | Shrikant Harne |
| collection | DOAJ |
| description | Summary: 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 cell helicity. However, the mechanism driving conformational changes in Fibril remains unknown. We expressed Fibril from S. citri in E. coli for its purification and characterization. Sodium dodecyl sulfate solubilized Fibril filaments and facilitated purification by affinity chromatography. An alternative protocol for obtaining enriched insoluble Fibril filaments was standardized using density gradient centrifugation. Electron microscopy of Fibril purified by these protocols revealed filament bundles. Probable domain boundaries of Fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of α-helical and β-sheet signatures in FT-IR measurements suggests that Fibril filaments consist of an assembly of folded globular domains, and not a β-strand-based aggregation like amyloid fibrils. |
| first_indexed | 2024-04-11T20:19:42Z |
| format | Article |
| id | doaj.art-dae12746da654b07b68743af5e02eb14 |
| institution | Directory Open Access Journal |
| issn | 2589-0042 |
| language | English |
| last_indexed | 2024-04-11T20:19:42Z |
| publishDate | 2022-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj.art-dae12746da654b07b68743af5e02eb142022-12-22T04:04:52ZengElsevieriScience2589-00422022-10-012510105055Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium SpiroplasmaShrikant Harne0Pananghat Gayathri1Indian Institute of Science Education and Research (IISER) Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, IndiaIndian Institute of Science Education and Research (IISER) Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, India; Corresponding authorSummary: 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 cell helicity. However, the mechanism driving conformational changes in Fibril remains unknown. We expressed Fibril from S. citri in E. coli for its purification and characterization. Sodium dodecyl sulfate solubilized Fibril filaments and facilitated purification by affinity chromatography. An alternative protocol for obtaining enriched insoluble Fibril filaments was standardized using density gradient centrifugation. Electron microscopy of Fibril purified by these protocols revealed filament bundles. Probable domain boundaries of Fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of α-helical and β-sheet signatures in FT-IR measurements suggests that Fibril filaments consist of an assembly of folded globular domains, and not a β-strand-based aggregation like amyloid fibrils.http://www.sciencedirect.com/science/article/pii/S258900422201327XBiochemistry methodsCell biologyProtein folding |
| spellingShingle | Shrikant Harne Pananghat Gayathri Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma iScience Biochemistry methods Cell biology Protein folding |
| title | Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma |
| title_full | Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma |
| title_fullStr | Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma |
| title_full_unstemmed | Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma |
| title_short | Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma |
| title_sort | characterization of heterologously expressed fibril a shape and motility determining cytoskeletal protein of the helical bacterium spiroplasma |
| topic | Biochemistry methods Cell biology Protein folding |
| url | http://www.sciencedirect.com/science/article/pii/S258900422201327X |
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