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.

Combination of Cα–H Hydrogen Bonds and van der Waals Packing Modulates the Stability of GxxxG-Mediated Dimers in Membranes

“…Structure-based analysis has been possible for a few structurally characterized dimeric systems, such as GpA 25 , 63 and BNIP3. 16 Conversely, large-scale comparative analyses, based either on combinatorial libraries, 23 , 64 − 68 comprehensive protein families, 69 or homology-based clusters of human proteins 53 have been performed primarily on sequences of unknown structure. Computational modeling has often been applied in coordination to these approaches to aid in the interpretation of experimental data.…”

BLaTM 2.0, a Genetic Tool Revealing Preferred Antiparallel Interaction of Transmembrane Helix 4 of the Dual-Topology Protein EmrE

“…S3 for an outline of the general workflow) 17 The GFP fluorescence of uninduced cells is subtracted from the measured data and the fluorescence signal per single cell is calculated, assuming that an OD 600 = 1.0 is equivalent to 8 * 10 8 cells/mL. Sigma-Aldrich) and a secondary anti-mouse-AP antibody (Promega).…”

“…In the BACTH system, interaction drives reconstitution of cAMP-producing adenylate cyclase, supporting reporter gene expression 14; 15; 16 . The most recent addition to this tool kit is BLaTM, where complementation of periplasmically located -lactamase fragments, that are genetically fused to interacting TMDs, confers ampicillin resistance to expressing cells 17 .…”

“…Since all required genetic components are plasmid-encoded, there is a wide choice of bacterial host strains with different features, such as varying lipid compositions of the inner membrane 31 . The BLaTM system can be used to investigate homotypic as well as heterotypic interactions since the TMDs are expressed within different hybrid proteins (N-BLa and C-BLa)17 . If measuring heterotypic interactions, potential homotypic associations should be determined in separate experiments, in order to determine the extent by which a heterotypic N-BLa/C-BLa interaction is competed by silent homotypic (N-BLa/N-BLa or C-BLa/C-BLa) associations that may occur, in parallel.…”

Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol

“…For construction of pBACOV-sfGFP, the sfGFP -gene was amplified using the primers sfGFP-fw and sfGFP-rv and the plasmid pET28A-sfGFP (kindly provided by Mark Teese) as template. The sfGFP coding sequence corresponds to the sequences published in [ 27 ]. pBACOV was linearized using the restriction endonucleases Mlu I and Xba I, thereby removing the coding sequence of the aprE signal peptide.…”