The overall objective is determination of the assembly and operation of the bacterial flagellar rotary motor. The research will enhance understanding of the molecular basis of motility and its regulation, chemiosmotic energy transduction and conformational switching in macromolecular assemblies. These fundamental issues are key to deciphering how living cells work. The understanding gained will be relevant, most immediately, for control of bacterial pathogens, and, given time, for cell based diagnoses of metabolism-related diseases. The rotor component of the Salmonella typhimurium flagellar motor has been over-produced in abundance by ho-expression of the four proteins (FIiF, FliG, FIiM, FIiN). Scanning transmission electron microscopy (STEM) will aim tc relate protein copies to subunit number through analysis of mass differences between complete and partial rotors containing a limited set of components. Cryoelectron microscopy (Cryo-EM) and metal replication will resolve subuni architecture and its change in mutant rotors locked in clockwise (CW) or counter-clockwise (CCW) rotating configurations. The number and location of CheY binding sites, will be determined utilizing high-density-gold labeled CheY, and related tc associated shifts in subunit tilt. More comprehensive 3D-reconstruction maps, in addition to analysis of the switching mechanism, should provide a basis for identification of the flagellar protein export apparatus and its interaction with the. motor machinery. Strains co-expressing the stator MotA, MotB proteins with the rotors have impaired growth, implying reconstitution of proton transport. Together with proton flux measurements, formation of intra-membrane particle rings will be monitored to define the role of FIIG and FIiM protein domains in docking, proton transport and, eventually, rotation. Mutation-induced changes in CheY-rotor affinity will be measured by evanescent wave fluorescence microscopy. These measurements will be made both in immobilized cells or envelopes, utilizing green fluorescent protein (GFP) or rhodamine.tagged CheY proteins respectively. They should provide novel information on the energetic basis for the high co-operativity of the flagellar motor switch, complementary to the planned structural studies.
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