title: Identifying ionic interactions within a membrane using BLaTM, a genetic tool to measure homo- and heterotypic transmembrane helix-helix interactions.
abstract: The assembly of integral membrane protein complexes is frequently supported by transmembrane domain (TMD) interactions. Here, we present the BLaTM assay that measures homotypic as well as heterotypic TMD-TMD interactions in a bacterial membrane. The system is based on complementation of β-lactamase fragments genetically fused to interacting TMDs, which confers ampicillin resistance to expressing cells. We validated BLaTM by showing that the assay faithfully reports known sequence-specific interactions of both types. In a practical application, we used BLaTM to screen a focussed combinatorial library for heterotypic interactions driven by electrostatic forces. The results reveal novel patterns of ionizable amino acids within the isolated TMD pairs. Those patterns indicate that formation of heterotypic TMD pairs is most efficiently supported by closely spaced ionizable residues of opposite charge. In addition, TMD heteromerization can apparently be driven by hydrogen bonding between basic or between acidic residues.
authors.affiliation: Munich Center For Integrated Protein Science (CIPSM) at the Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Schanzenbach
authors.affiliation: Munich Center For Integrated Protein Science (CIPSM) at the Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors.forename: Fabian C
authors.initials: FC
authors.lastname: Schmidt
authors.affiliation: Munich Center For Integrated Protein Science (CIPSM) at the Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Breckner
authors.affiliation: Munich Center For Integrated Protein Science (CIPSM) at the Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors.forename: Mark G
authors.initials: MG
authors.lastname: Teese
authors.affiliation: Munich Center For Integrated Protein Science (CIPSM) at the Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Langosch
mesh_terms: D000595:Amino Acid Sequence; D000667:Ampicillin / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006021:Glycophorins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; C409972:GYPA protein, human; D006021:Glycophorins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D000667:Ampicillin; D050862:Oxidoreductases Acting on Sulfur Group Donors; C481160:QSOX2 protein, human; D001618:beta-Lactamases