Detection of Ring Structure and Its Flexibility in Periplasmic Region of FliL, Bacterial Flagellar Stator-Associate...

FliL is a conserved single-transmembrane protein implicated in stator function and load adaptation of bacterial flagellar motors. We analyzed the oligomeric organization and structural plasticity of the periplasmic domain of polar-flagellum FliL from Vibrio alginolyticus (^pofFliL_C) and compared it with detergent-solubilized full-length ^pofFliL. Recombinant proteins were purified by affinity and size-exclusion chromatography (SEC). SEC profiles indicated condition-dependent assemblies; ^pofFliL_C eluted broadly at ~100 kDa and partially shifted to ~45 kDa on re-chromatography, whereas full-length ^pofFliL eluted as large, heterogeneous assemblies (~180 kDa). Negative-stain electron microscopy (EM) revealed abundant ~10-nm rings for ^pofFliL_C but heterogeneous particles for full-length protein. Cryo-EM single-particle analysis of the ^pofFliL_C yielded high-quality two-dimensional top/bottom-view class averages that show ring particles with clear subunit repeats; class averages correspond to apparent 10-, 11- and 12-fold stoichiometries, but severe preferred orientation and sample heterogeneity prevented reliable three-dimensional reconstruction. High-speed AFM imaged ~15 nm rings and captured transient subunit rearrangements and opening/closing events; representative AFM frames can be interpreted as 10-subunit-like, yet AFM contrast and tip convolution limited unambiguous subunit counting across the population. These complementary data indicate that the ^pofFliL_C SPFH-like periplasmic domain forms flexible, dynamic ring oligomers whose stoichiometry and conformation are modulated by membrane association and experimental conditions. We propose that such plasticity enables FliL to act as an adaptable scaffold for stator engagement and mechanosensitive remodeling of the motor.