Evidence for the Supramolecular Organization of a Bacterial Outer-Membrane Protein from In Vivo Pulse Electron Paramagnetic Resonance Spectroscopy

Abstract

In the outer membrane of Gram-negative bacteria, membrane proteins are thought to be organized into domains or islands that play a role in the segregation, movement, and turnover of membrane components. However, there is presently limited information on the structure of these domains or the molecular interactions that mediate domain formation. In the present work, the Escherichia coli outer membrane vitamin B 12 transporter, BtuB, was spin-labeled, and double electron−electron resonance was used to measure the distances between proteins in intact cells. These data together with Monte Carlo simulations provide evidence for the presence of specific intermolecular contacts between BtuB monomers that could drive the formation of string-like oligomers. Moreover, the EPR data provide evidence for the location of the interacting interfaces and indicate that lipopolysaccharide mediates the contacts between BtuB monomers.

In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels

“…Membrane proteins are expressed at a very low level (ranging between 10 2 –10 5 copies per cell) and to observe them close to the native expression level, very sensitive spin labels and labeling strategies need to be developed. Also, several membrane proteins form homo‐ or heterooligomeric complexes [14] and orthogonal labeling strategies would greatly facilitate their investigations. The carbon‐centered trityl radicals have attained much attention for ESR and also as an orthogonal tag with other spin labels [8, 15–22] .…”

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

“…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 B 12 (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).…”

“…Glycerol stocks of dsbA - cells expressing L63C-T188C, S65C-T188C, N72C-T188C, V90C-T188C, S93C-T188C and V90C-S237C BtuB with and without R14A, V90C-T188C-D316A and V90C-T188C-R14A-D316A BtuB were used to directly inoculate the pre-precultures (Luria Bertani media with 200 µg/ml ampicillin), grown for 8 hours at 37°C and used to inoculate the MM precultures. The main MM cultures were inoculated with precultures, grown until OD 600 ∼0.3 and spin labeled (33). Briefly, the cells were spin labeled with methanethiosulfonate spin label (MTSSL) ((1-oxy-2,2,5,5-tetramethylpyrrolinyl-3-methyl)methanethiosulfonate), (Cayman Chemical, Ann Arbor Michigan) in 100 mM HEPES buffer (pH 7.0) containing 2.5% (w/v) glucose with the final concentration of 7.5 nmol/ ml of cell culture at OD600 0.3 for 30 min at room temperature (RT).…”

“…MM main cultures of V90C-T188C and S93C-T188C with and without R14A were grown for 8 hours at 37°C. The harvested cells were used to isolate intact OM (33). After the second spin at 118,370 × g for 60 min at 4°C the pellets were resuspended in 5 mL of HEPES buffer.…”

From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

“…However, up to now, spin labelling techniques targeting membrane proteins in cellular environments were successfully applied only to outer membrane proteins of Escherichia coli [112][113][114][115], but methods to label a membrane protein in the inner membrane of E. coli or in the cellular membranes of mammalian cells have not yet been established.…”