Disulfide Chaperone Knockouts Facilitate Double Spin Labelling of an Outer Membrane Transporter For in Vitro EPR Studies

Abstract

The outer membrane (OM) of Gram-negative bacteria contains a number of porins and transporters that are crucial for bacterial growth and survival. These transporters include TonB-dependent transporters (TBDTs) that facilitate the active uptake of trace metals and complex carbohydrates, and are critical for the microbiome as well as the survival and virulence of many pathogens. During active transport, TBDTs couple with the trans-periplasmic protein, TonB and utilize energy from the inner membrane (IM) proton motive force. However, the exact mechanism of transport is not well understood. Recently, we have shown that it is possible to examine the structure of BtuB, the E. coli vitamin B12 transporter, using Electron Paramagnetic Resonance (EPR) spectroscopy in intact cells. Distance measurements using pulse EPR require that pairs of labels be placed within BtuB, which was accomplished by site-directed spin labelling of cysteines. in cells, this required the use of a knockout of the Dsb system that functions to oxidize pairs or cysteines. Using a dsbA- strain, we have shown that the protein appears to have a different configuration in vivo when compared to earlier work of reconstituted BtuB in liposomes. While comparing the in vivo and in vitro systems, we discovered that cysteine double mutants’ in vitro labeling efficiencies are poor when expressed in a wild-type strain. in contrast, we were able to efficiently spin label purified BtuB expressed in dsbA- strain. These results indicate that knocking out the Dsb system also promotes efficient double labeling for reconstituted membrane proteins that are isolated from the OM and facilitate the studies of BtuB in both in vivo and in vitro conditions.