Structure-function studies of self-assembling flagellin proteins from Salmonella typhimurium and Aquifex pyrophilus.

摘要

The eubacterial flagellum is a complex structure with an elongated extracellular filament that is primarily composed of a single protein termed flagellin. The highly conserved N- and C-termini of flagellin are important in its export and assembly, whereas the middle "hypervariable" region is highly variable in size across different species and is largely dispensable. In Salmonella typhimurium phase 1 flagellin (FliC), this hypervariable region encodes two solvent-exposed D2 and D3 domains that form a knob-like feature on flagella fiber, with unknown function. The structural and physiological role of the hypervariable region in flagellar assembly, stability and cellular motility was investigated. A library of random internal deletion variants of FliC was constructed and screened for functional variants using a swarming agar motility assay. The relative cellular motility and propulsive force of ten representative functional variants were determined in semi-solid and liquid media using swarming motility assays, video microscopy, and optical trapping of single cells. All ten variants investigated exhibited diminished motility and mechanical stability, with partial motility observed for internal deletions less than 75 residues and nearly complete loss of motility for deletions greater than 100 residues. Homology modeling predicted disruptions of secondary structures in each variant. The hypervariable region middle domain appears to have an important role in stabilizing the folded conformation of the flagellin protein and resulting mechanical stability and propulsive force of the flagella fibers. A high-throughput motility assay was developed and used to screen S. typhimurium against a compound library for potential antibiotics. A structure-function study was performed on the predicted role of a D1 domain hairpin turn loop region as a conformational switch. The feasibility of engineering novel metal binding sites into flagellin was also examined via site-directed mutagenesis. A predictive 3-D structure of a thermostable flagellin protein (FlaA) from Aquifex pyrophilus was developed, using the S. typhimurium flagellin as a template, and analyzed with respect to determinants of thermostability. The predominant stabilizing factors in FlaA appear to be increased hydrophobicity, fewer intramolecular ion pairs, an increase in the positive charge potential of the FlaA D0 and D1 domains, and increased intermolecular salt bridges.