In vitro characterization of FlgB, FlgC, FlgF, FlgG, and FliE, flagellar basal body proteins of Salmonella.
The bacterial flagellar basal body is a rotary motor. It spans the cytoplasmic and outer membranes and drives rapid rotation of a long helical filament in the cell exterior. The flagellar rod at its central axis is a drive shaft that transmits torque through the hook to the filament to propel the bacterial locomotion. To study the structure of the rod in detail, we have established purification procedures for Salmonella rod proteins, FlgB, FlgC, FlgF, FlgG, and also for FliE, a rod adapter protein, from an Escherichia coli expression system. While FlgF was highly soluble, FlgB, FlgC, FlgG and FliE tended to self or cross-aggregate into fibrils in solutions at neutral pH or below, at high ionic strength, or at high protein concentration. These aggregates were characterized to be beta-amyloid fibrils, unrelated to the rod structure formed in vivo. Under non-aggregative conditions, no protein-protein interactions were detected between any pairs of these five proteins, suggesting that their spontaneous, template-free polymerization is strongly suppressed. Limited proteolyses showed that FlgF and FlgG have natively unfolded N and C-terminal regions of about 100 residues in total just as flagellin does, whereas FlgB, FlgC and FliE, which are little over 100 residues long, are unfolded in their entire peptide chains. These results together with other data indicate that all of the ten flagellar axial proteins share structural characteristics and folding dynamics in relation to the mechanism of their self-assembly into the flagellar axial structure.
Saijo-Hamano
Yumiko
Y
Protomic NanoMachine Project, ERATO, JST, Seika, Kyoto, Japan.
Uchida
Naoko
N
Namba
Keiichi
K
Oosawa
Kenji
K
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D005407:Flagella / Q000737:chemistry*; D006868:Hydrolysis; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D012475:Salmonella / Q000737:chemistry*
D001426:Bacterial Proteins; D017931:DNA Primers