The bacterial flagellar motor (BFM) is an 11 MDa superstructure that powers the rotation of long, helical flagellar filaments, allowing bacteria to swim through viscous media1 . A 45 nm cytosolic protein ring (C-ring) known as the switch complex, forms the heart of the BFM as it can convert cation flux into mechanical rotation up to speeds of 1700 Hz and can make the motor change speed, stop and change direction in milliseconds in response to its chemical environment2 3 4 . The switch complex is composed of dozens of copies of proteins, FliG, FliM and FliN, which assemble into a series of stacked rings on top of a transmembrane structural scaffold (MS-ring), which is composed of multiple copies of the protein, FliF.
Due to being a large, highly dynamic membrane protein complex, which cannot be readily dissembled or manipulated, there are currently a limited number of ways of studying the structure and function of its various subunits at the atomic level. Currently no-one has been able to provide a complete picture of the BFM at the atomic level, nor are the kinetics of its assembly are understood.
We present a novel method of studying the BFM switch complex - to build it artificially using DNA-based nanoscaffolds, replacing the transmembrane MS-ring. The scaffold is functionalised with trisNi-NTA to initiate assembly of a histidine-tagged FliG ring, from which FliM and FliN rings can then assemble. Using this method has allowed us to monitor the assembly of the FliG ring using Bio-Layer Interferometry (BLI), which can provide the kinetics and stability of assembly in real-time. We believe that FliG ring assembly is cooperative, where a structural scaffold is required for the nucleation of FliG monomers to initiate oligomerisation of the FliG ring. Details of these kinetic studies will be reported.