Key amino acid residues involved in the transitions of L- to R-type protofilaments of the Salmonella flagellar filament.

The flagellar filament enables bacteria to swim by functioning as a helical propeller. The filament is a supercoiled assembly of a single protein, flagellin, and is formed by 11 protofilaments arranged in a circle. Bacterial swimming and tumbling correlate with changes of the various helical structures, called polymorphic transformation, that are determined by the ratios of two distinct forms of protofilaments, the L and R types. The polymorphic transformation is caused by transition of the protofilament between L and R types. Elucidation of this transition mechanism has been addressed by comparing the atomic structures of L- and R-type straight filaments or using massive molecular dynamic simulation. Here, we found amino acid residues required for the transition of the protofilament using fliC-intragenic suppressor analysis. We isolated a number of revertants producing supercoiled filaments from mutants with straight filaments and identified the second-site mutations in all of the revertants. The results suggest that Asp107, Gly426, and Ser448 and Ser106, Ala416, Ala427, and Arg431 are the key residues involved in inducing supercoiled filaments from the R- and the L-type straight filaments, respectively. Considering the structures of the R- and L-type protofilaments and the relationship between the rotation of the flagellar motor and the polymorphic transformation, we propose that Gly426, Ala427, and Arg431 contribute to the first stage of the transition and that Ser106, Asp107, and Ala416 play a role in propagating the transitions along the flagellar filament.

Synthesis of uniform and dispersive calcium carbonate nanoparticles in a protein cage through control of electrostatic potential.

We have synthesized calcium carbonate nanoparticles (Ca-NPs) in the cavity of a cage-shaped protein, apoferritin, by regulating the electrostatic potential of the molecule. The electrostatic potential in the cavity was controlled by pH changes resulting from changes in the dissolved carbon dioxide (CO(2)) concentration in the reaction solution. Recombinant L-apoferritin was mixed with a suspension of calcium carbonate (CaCO(3)), and the mixture was pressurized with gaseous CO(2) at 2 MPa. The pH of the solution decreased from 9.3 to 4.4; the CaCO(3) dissolved during pressurization, and then precipitated after the pressure was reduced to ambient. After repeating the pressurization/depressurization process three times, about 70% of the apoferritin molecules were found to contain nanoparticles with an average diameter of 5.8 ± 1.2 nm in their cavity. Energy-dispersive X-ray spectroscopy and electron diffraction analysis showed that the nanoparticles were calcite, one of the most stable crystal forms of CaCO(3). Electrostatic potential calculations revealed a transition in the potential in the apoferritin cavity, from negative to positive, below pH 4.4. The electrostatic potential change because of the change in pH was crucial for ion accumulation. Since the Ca-NPs synthesized by this method were coated with a protein shell, the particles were stably dispersed in solution and did not form aggregates. These Ca-NPs may be useful for medical applications such as synthetic bone scaffolds.