Solubilization and purification of the MotA/MotB complex of Escherichia coli

Abstract

Bacterial flagella are driven at their base by a rotary motor fueled by the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which function together to conduct ions across the membrane and couple ion flow to rotation. An invariant aspartate residue in MotB (Asp32 in the protein of E. coli) is essential for rotation and appears to have a direct role in proton conduction. A recent study showed that changes at Asp32 in MotB cause a conformational change in the complex, as evidenced by altered patterns of protease susceptibility of MotA [Kojima, S., and Blair, D. F. (2001) Biochemistry 40 (43), 13041-13050]. It was proposed that protonation/deprotonation of Asp32 might regulate a conformational change in the stator that acts as the powerstroke to drive rotation of the rotor. Biochemical studies of the MotA/MotB complex have been hampered by the absence of a suitable assay for its integrity in detergent solution. Here, we have studied the behavior of the MotA/MotB complex in a variety of detergents, making use of the protease-susceptibility assay to monitor its integrity. Among about 25 detergents tested, a few were found to solubilize the proteins effectively while preserving certain conformational properties characteristic of an intact complex. The detergent dodecylphosphocholine, or DPC, proved especially effective. MotA/MotB complexes purified in DPC migrate with an apparent size of approximately 300 kDa in gel-filtration columns, and retain the Asp32-modulated conformational differences seen in membranes. (35)S-radiolabeling showed that MotA and MotB are present in a 2:1 ratio in the complex. Purified MotA/MotB complexes should enable in vitro study of the proton-induced conformational change and other aspects of stator function.