Templated self-assembly of the bacterial flagellar motor torque ring in vitro

Thesis

The Bacterial Flagellar Motor is an ion-driven rotary motor employed by many bacteria for motility and surface sensing, and a model system for the self-assembly and regulation of large protein complexes.

Early in motor biogenesis a ring of the integral membrane protein FliF templates the cytoplasmic assembly of FliG, which subsequently templates transient incorporation of FliM₁:FliN₃ subunits. FliG, FliM and FliN collectively comprise the C-ring: site of torque generation and directional switching. Switching is regulated by binding of CheY-P to FliM₁:FliN₃, propagating a co-operative conformational change throughout the entire C-ring. The molecular details of C-ring structure, assembly and dynamics are unclear.

Averaged cryo-EM structures of purified motors feature a curious symmetry mismatch between the FliF ring (~26-fold) and C-ring (~34-fold), while in vivo fluorescence shows a variation in FliM₁:FliN₃ population with rotation direction that is not resolved in the EM structures. The stoichiometries of FliG/M/N are all disputed, and cannot yet be measured accurately in vivo. More powerful techniques are available in vitro, but it is unclear whether dynamic structural features of the motor survive purification.

Therefore, we aspire to assemble a C-ring in vitro as a platform for in vitro study. Furthermore, by substituting the difficult-to-reconstitute FliF template with a controllable DNA-origami mimic, we envisage studying self-assembly from the bottom up through template remodelling: a novel concept in the study of protein self-assembly.

This thesis describes the development of linear DNA templates mimicking short fragments of the FliF ring. FliG arrangement on these templates is specified by DNA sequence design, and can be quantified with single-molecule fluorescence and native gel electrophoresis. The methods developed here will allow testing of the FliG domain-swap polymerization model as a mechanistic explanation for the symmetry mismatch, and lay the foundations for in vitro construction of a complete C-ring.

Authors: Tusk, S

• DOI: 10.5287/ora-gjza9e7ey
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Bacterial Flagella Motor; DNA nanotechnology
• Subjects: Synthetic biology; Single-molecule Biophysics; Biochemistry; Nanotechnology