Dataset Information

Bacterial outer membrane channel for divalent metal ion acquisition.

ABSTRACT: The prevailing model of bacterial membrane function predicts that the outer membrane is permeable to most small solutes because of pores with limited selectivity based primarily on size. Here, we identified mnoP in the Gram-negative bacterium Bradyrhizobium japonicum as a gene coregulated with the inner membrane Mn(2+) transporter gene mntH. MnoP is an outer membrane protein expressed specifically under manganese limitation. MnoP acts as a channel to facilitate the tranlocation of Mn(2+), but not Co(2+) or Cu(2+), into reconstituted proteoliposomes. An mnoP mutant is defective in high-affinity Mn(2+) transport into cells and has a severe growth phenotype under manganese limitation. We suggest that the outer membrane is a barrier to divalent metal ions that requires a selective channel to meet the nutritional needs of the cell.

SUBMITTER: Hohle TH

PROVIDER: S-EPMC3174606 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Bacterial outer membrane channel for divalent metal ion acquisition.

Hohle Thomas H TH Franck William L WL Stacey Gary G O'Brian Mark R MR

Proceedings of the National Academy of Sciences of the United States of America 20110831 37

The prevailing model of bacterial membrane function predicts that the outer membrane is permeable to most small solutes because of pores with limited selectivity based primarily on size. Here, we identified mnoP in the Gram-negative bacterium Bradyrhizobium japonicum as a gene coregulated with the inner membrane Mn(2+) transporter gene mntH. MnoP is an outer membrane protein expressed specifically under manganese limitation. MnoP acts as a channel to facilitate the tranlocation of Mn(2+), but no ...[more]

PMID: 21880957

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Project description:Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

Dataset Information

Bacterial outer membrane channel for divalent metal ion acquisition.

Publications

Bacterial outer membrane channel for divalent metal ion acquisition.

Similar Datasets

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