Structural, Biochemical And Biophysical Studies Of The Bacterial Flagellar Switch Complex
Many bacteria use flagella operated by rotary motors to swim. These complex structures contain more than 25 different proteins that self assemble to generate torque and regulate the sense of flagellar rotation. A key molecular event during chemotaxis is the interaction between the phosphorylated response regulator CheY (CheY-P) and the flagellar switch complex, which serves to switch the direction of flagellar rotation between clockwise and counterclockwise, in to order tumble or swim smoothly, respectively. The flagellar switch complex, composed of FliM, FliG and FliN, is responsible for the changes in the direction of rotation of the flagella, torque generation and flagellar assembly. FliM is the switch complex component that interacts with CheY-P and with the other two components of the switch complex and it is known to be important for flagellar assembly. FliG is known to interact with the motor complexes MotAB, which provide the energy necessary for torque generation. However, the interaction FliG-FliM is not primarily involved in flagellar assembly or torque generation but instead might play a critical role in switching. To understand the mechanism of flagellar switching and its relationship to torque generation and signal amplification, I have cloned, expressed, purified, characterized and crystallized for the first time a two-component flagellar switch complex FliM/FliG. The structure is in agreement with biochemical and mutational experiments in terms of interaction interface between FliG and FliM. Also, the structure shows an interesting conformation of FliG middle domain that is different to the one previously reported. A FliM dimer is reported and extensive biophysical studies have being performed to try to understand FliG - mediated FliM self-assembly and how relevant it is to switching. Our crystal structure and biochemical studies provide new insights into a more complete model for the molecular mechanism of flagellar motor switching.
dissertation or thesis