Deducing the organization of a transmembrane domain by disulfide cross-linking. The bacterial chemoreceptor Trg.
The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four segments, two from each subunit of the homodimer. We used site-specific mutagenesis to introduce cysteines into those segments and oxidative cross-linking of cysteine pairs to identify residues that are near each other in space. Propensity for cross-linking was determined for pairs of homologously placed cysteines in the two subunits of the dimer at all 54 possible positions. Also, combinations of cysteines were identified that readily oxidized to join heterologous segments within or between monomers. These patterns of cross-linking were used to develop a model for the three-dimensional structure of the transmembrane domain in which the four transmembrane segments are helices associated in a bundle, with stronger interactions near the periplasm and weaker interactions near the cytoplasm. The striking similarity of this 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 analysis of patterns of disulfide cross-linking is sufficient to deduce a detailed three-dimensional structure for a transmembrane domain.
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
G F
GF
Lee
G G
GG
Burrows
M R
MR
Lebert
D P
DP
Dutton
G L
GL
Hazelbauer
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D002628:Chemoreceptor Cells / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C041476:Trg protein, E coli