title: Identification of functionally important helical faces in transmembrane segments by scanning mutagenesis.
abstract: We applied mutational analysis to a protein domain that functions in neither catalysis nor binding but, rather, in transmembrane signaling. The domain is part of chemoreceptor Trg from Escherichia coli. It contains four transmembrane segments, two from each subunit of the homodimer. We used cysteine scanning to investigate the functional importance of each of 54 residues in the two transmembrane segments. Cysteines at some positions resulted in subtle but significant reductions in tactic response. Those positions defined a specific helical face on each segment, implying that the segments function as helices. The functionally important faces corresponded to structural, helical packing faces identified independently by biochemical studies. All functionally impaired receptors exhibited altered signaling properties, either reduced signaling upon stimulation or induced signaling in the absence of stimulation. The distribution of substitutions creating these two phenotypes implied that conformational signaling involves movement between the two transmembrane helices within a subunit and that signaling is optimal when stable interactions are maintained across the interface between subunits.
authors.affiliation: Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
authors.forename: G F
authors.initials: GF
authors.lastname: Lee
authors.forename: D P
authors.initials: DP
authors.lastname: Dutton
authors.forename: G L
authors.initials: GL
authors.lastname: Hazelbauer
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis; D003545:Cysteine / Q000032:analysis; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D011487:Protein Conformation; D015398:Signal Transduction
chemical_list: D008565:Membrane Proteins; D003545:Cysteine