Disulfide Chaperone Knockouts Enable In Vivo Double Spin Labeling of an Outer Membrane Transporter

Abstract

Recent advances in the application of electron paramagnetic resonance spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer membrane (OM) proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli OM 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 periplasmicfacing 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 OMs or reconstituted systems, and the ability to label and perform electron paramagnetic resonance in cells is expected to be applicable to a range of other bacterial OM proteins.

Structural intermediates observed only in intactEscherichia coliindicate a mechanism for TonB-dependent transport

“…1a). These included multiple sites on the extracellular loops of BtuB (18)(19)(20) as well as two sites in the core region (18).…”

“…Recent work has also shown that the efficient incorporation of pairs of spin labels to make distance measurements using DEER required the use of a strain deficient in the disulfide bond formation (Dsb) chaperone system (18). We tested several additional single cysteine mutants in BtuB using a DsbA - strain to determine whether spin labeling of additional sites in the core was possible.…”

“…Whole cells sample Preparation: dsbAcells expressing V90C-T188C, L63C, S65C, N72C, V90C and S93C BtuB were grown in minimal media (MM) supplemented with 200 µg/ml ampicillin, 0.2 % w/v glucose, 150 µM thiamine, 3 mM MgSO4, 300 µM CaCl2, 0.01 % w/v methionine and 0.01% w/v arginine (18). Cells expressing BtuB with the V90C-T188C mutation was spin labeled as described (18) and the aliquots of processed cell pellets were mixed with vitamin B12 (0, 1, 5, 20, 30, 60 and 100 µM final concentrations). The cells expressing L63C, S65C, N72C, V90C and S93C BtuB mutants were processed as described in Nyenhuis et al (33).…”

“…Since transport in this family of transporters has never been reconstituted, it has never been established that the isolated, purified, and membrane reconstituted BtuB is capable of transport. Recently, we developed an approach to attach spin labels to either extracellular or periplasmic sites on BtuB in intact cells, thereby permitting EPR measurements to be made under conditions where the protein is known to be functional (17, 18). Preliminary measurements made on BtuB indicate that it behaves differently in the intact cell than it does in a purified reconstituted phospholipid system.…”

“…Preliminary measurements made on BtuB indicate that it behaves differently in the intact cell than it does in a purified reconstituted phospholipid system. For example, a substrate-dependent change in the core domain of BtuB involving SB3 is observed in situ but is not seen in a detergent treated OM preparation (18). Moreover, the extracellular loops of BtuB are also highly constrained in the intact cell, and substrate-induced structural changes and structural heterogeneity that is observed for BtuB in proteoliposomes is not observed for BtuB in situ (19).…”