Figure 1.

Mapping interfacial residue positions of the oligo-leucine TMS (leu20) by asparagine-scanning mutagenesis in membranes. (A) Self-assembly of leu20 and its asparagine mutants as determined by the ToxR system. Mutants with strongly increased β-galactosidase activities (mean ± SE, n = 12) relative to the parental leu20 sequence follow a heptad repeat pattern and are labeled accordingly. (B) Protein expression levels were comparable as confirmed by Western blot. The order of samples corresponds to that in A. (C) Growth kinetics of PD28 cells expressing the ToxR constructs in minimal medium with maltose were similar, thus indicating similar concentrations of the different ToxR proteins in the membrane. A construct in which the TMS had been deleted (ΔTM) served as negative control. (D) Alignment of leu20 to the heptad pattern revealed by asparagine-scanning mutagenesis. The helical wheel diagram depicts how a and d positions form the interface of a leucine zipper. Although a pair of helices is shown for the sake of simplicity, the same geometry of side-chain packing applies to complexes with more than two helices.