Purification of the Na+-Driven PomAB Stator Complex and Its Analysis Using ATR-FTIR Spectroscopy.

Abstract

The flagellar motor of marine Vibrio is driven by the sodium-motive force across the inner membrane. The stator complex, consisting of two membrane proteins PomA and PomB, is responsible for energy conversion in the motor. To understand the coupling of the Na+ flux with torque generation, it is essential to clearly identify the Na+-binding sites and the Na+ flux pathway through the stator channel. Although residues essential for Na+ flux have been identified by using mutational analysis, it has been difficult to observe Na+ binding to the PomAB stator complex. Here we describe a method to monitor the binding of Na+ to purified PomAB stator complex using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. This method demonstrates that Na+-binding sites are formed by critical aspartic acid and threonine residues located in the transmembrane segments of PomAB.