DEDUCING THE ORGANIZATION OF A TRANSMEMBRANE DOMAIN BY DISULFIDE CROSS-LINKING - THE BACTERIAL CHEMORECEPTOR TRG

The transmembrane domain of chemoreceptor Trg from Escherichia coli co ntains four segments, two from each subunit of the homodimer. me used site specific mutagenesis to introduce cysteines into those segments a nd oxidative cross-linking of cysteine pairs to identify residues that are near each other in space. Propensity for cross-linking was determ ined for pairs of homologously placed cysteines in the two subunits of the dimer at all 54 possible positions. Also, combinations of cystein es were identified that readily oxidized to join heterologous segments within or between monomers. These patterns of cross-licking were used to develop a model for the three-dimensional structure of the transme mbrane domain in which the four transmembrane segments are helices ass ociated in a bundle, with stronger interactions near the periplasm and weaker interactions near the cytoplasm. The striking similarity of th is model to a model for the transmembrane domain of chemoreceptor Tar, derived using the same experimental strategy, strengthens the notion that a combination of comprehensive cysteine substitutions and analysi s of patterns of disulfide cross-licking is sufficient to deduce a det ailed three-dimensional structure for a transmembrane domain.