Disulfide chaperone knock-outs enable in-vivo double spin-labeling of an outer-membrane transporter

Abstract

Recent advances in the application of EPR spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer-membrane proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli outer-membrane vitamin B 12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated outer membranes or reconstituted systems, and the ability to label and perform EPR in cells is expected to be applicable to a range of other bacterial outer-membrane proteins.