Role of the disordered terminal regions of flagellin in filament formation and stability.
Terminal regions of flagellin from Salmonella typhimurium, residues 1 to 65 and 451 to 494, have no ordered tertiary structure in solution, which makes them very susceptible to proteolytic degradation. Flagellin was subjected to mild controlled proteolytic treatment with highly specific proteases to remove terminal segments from the disordered regions. It is demonstrated here that various fragments can be readily prepared that differ from each other in 1 x 10(3) to 2 x 10(3) Mr segments in their NH2- or COOH-terminal regions. Terminally deleted fragments of flagellin were used to clarify the role of the disordered regions in the self-assembly of flagellin. The polymerization ability of the fragments was tested by inducing filament formation with ammonium sulfate. We found that fragments of flagellin containing large terminal deletions could form straight filaments, although the stability of these filaments required high salt concentrations. Even a fragment lacking the whole mobile COOH-terminal part of flagellin and 36 residues from the NH2-terminal region could form long filaments. The fragments could be also polymerized onto native flagellar seeds, suggesting that the subunit packing of the filaments of fragments is similar to that of the native ones. The fragments could also copolymerize with native flagellin, resulting in various helical forms. Filaments of fragments were found to be straight at both pH 4.0 and pH 12.5, indicating that they might have lost their polymorphic ability. Our results show that the major part of the disordered terminal regions of flagellin is not essential for polymerization, but it does play an important role in stabilization of the filaments and in influencing their polymorphic conformation.
ERATO, Molecular Dynamic Assembly Project, Tsukuba, Japan.
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Vonderviszt
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Aizawa
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Namba
D000645:Ammonium Sulfate / Q000378:metabolism; D002268:Carboxypeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005407:Flagella / Q000737:chemistry / Q000378:metabolism*; D005408:Flagellin / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D011108:Polymers / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism; D014961:X-Ray Diffraction
D046911:Macromolecular Substances; D011108:Polymers; D005408:Flagellin; D002268:Carboxypeptidases; D010450:Endopeptidases; D000645:Ammonium Sulfate