MUTATIONAL ANALYSIS OF A TRANSMEMBRANE SEGMENT IN A BACTERIAL CHEMORECEPTOR

Trg is a member of a family of receptors that mediates chemotaxis by E scherichia coli, Its transmembrane domain is a loose four-helix bundle consisting of two helices from each of the two identical subunits, Th is domain mediates transmembrane signaling through a conformational ch ange in which the second transmembrane segment (TM2) is thought to mov e relative to TM1, but mutational analysis of TM2 by cysteine scanning had identified only a few positions at which substitutions perturbed function or induced signaling, Thus, rye performed mutational analysis by random mutagenesis and screening, Among 42 single-residue substitu tions in TM2 that detectably altered function, 16 had drastic effects on receptor activity, These substitutions defined a helical face of TM 2. This functionally important surface was directed into the protein i nterior of the transmembrane domain, where TM2 faces the helices of th e other subunit. The functionally perturbing substitutions did not app ear to cause general disruption of receptor structure but rather had m ore specific effects, altering aspects of transmembrane signaling, An in vivo assay of signaling identified some substitutions that reduced and others that induced signaling. These two classes were distributed along adjacent helical faces in a pattern that strongly supports the n otion that conformational signaling involves movement between TM2 and TM1 and that signaling is optimal when stable interactions are maintai ned across the interface between the homologous helices in the transme mbrane domain, Our mutational analysis also revealed a striking tolera nce of the chemoreceptor for substitutions, including charged residues , usually considered to be disruptive of transmembrane segments.