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 of54 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.Four transmembrane receptors mediate chemotaxis by Escherichia coli (1-3). Recognition of positive stimuli generates counterclockwise (CCW) rotation of bacterial flagella, which in turn results in coordinated swimming. Recognition of negative stimuli generates clockwise (CW) rotation and uncoordinated tumbles. Chemoreceptor Trg recognizes sugaroccupied galactose-and ribose-binding proteins and thereby mediates chemotactic response to those sugars. Chemoreceptors are homodimers (4-6). Receptor monomers, "60 kDa, have a transmembrane domain consisting of two hydrophobic segments, a periplasmic, ligand-binding domain and a highly conserved cytoplasmic domain that mediates intracellular signaling by controlling a noncovalently associated protein kinase and that mediates adaptation through dynamic methylation and demethylation of several glutamyl residues. An understanding of transmembrane signaling in chemoreceptors of E. coli should be relevant not only to methyl-accepting taxis proteins in other bacteria (7) but also to the many related environmental sensor proteins in prokaryotes (1, 3) and eukaryotes (8).The monomer-dimer equilibrium for chemoreceptors strongly favors dimers and does not appear to shift in the course of the sensory cycle (4), implying that transmembrane signaling involves conformational changes within the dimer. It is not known what features of the domain are important for or involved in transmembrane signaling. We approached these issues with mutational analysis by "cysteine scanning." The approach has many of the features and advantages of "alaninescanning mutagenesis" (9). Cysteine is an attractive choice for scanning mutagenesis of transmembrane segments because itsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisemen...

Myxococcus xanthus is a gliding bacterium that possesses two motility systems, the adventurous (A-motility) and social (S-motility) systems. A-motility is used for individual cell gliding, while S-motility is used for gliding in multicellular groups. Video microscopy studies showed that nla24 cells are non-motile on agar surfaces, suggesting that the nla24 gene product is absolutely required for both A-motility and S-motility under these assay conditions. S-motility requires functional type IV pili, wild-type LPS O-antigen, and an extracellular matrix of exopolysaccharide (EPS) and protein called fibrils. The results of expression studies and tethering assays indicate that the nla24 mutant has functional type IV pili. The nla24 mutant also produces wild-type LPS. However, several lines of evidence suggest that the nla24 mutant is defective for production of the EPS portion of the fibril matrix. The nla24 mutant is also defective for transcription of two genes (aglU and cglB) known to be required for A-motility, which is consistent with the idea that nla24 cells are defective for A-motility. Based on these findings, it is proposed that the putative transcriptional activator Nla24 regulates a subset of genes that are important for A-motility and S-motility in M. xanthus.

Chemotactic transducer proteins of Escherichia coli contain four or five methyl-accepting glutamates that are crucial for sensory adaptation and gradient sensing. Two residues arise from posttranslational deamidation of glutamines to yield methyl-accepting glutamates. We addressed the significance of this arrangement by creating two mutated trg genes: trg(SE), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(SQ), in which all five were glutamines. We found that the normal (3E,2Q) configuration was not an absolute requirement for synthesis, assembly, or stable maintenance of transducers. Both mutant proteins were methylated, although Trg(5Q) had a reduced number of methyl-accepting sites because two glutamines at adjacent residues were blocked for deamidation and thus could not become methyl-accepting glutamates. The glutamine-glutamate balance had striking effects on signaling state. Trg(5E) was in a strong counterclockwise signaling configuration, and Trg(5Q) was in a strong clockwise configuration. Interestingly, amides were not as effective as methyl esters in balancing counterclockwise signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(SE) or trg(5Q) exhibited reduced effectiveness, probably reflecting perturbations of the counterclockwise/clockwise ratio caused by newly synthesized transducers not modified rapidly enough to produce a balanced signaling state during growth. These defects were evident for cells in which other transducers were not available to contribute to balanced signaling or were present at lower levels than the mutant proteins.Methyl-accepting transducers are sophisticated chemoreceptor proteins designed to detect temporal gradients by comparing measurements of current concentration to a record of concentrations in the recent past (for a recent review, see reference 9). In Escherichia coli there are four different transducers, each a transmembrane receptor for a particular set of compounds. Transducers are organized into two hydrophilic domains, one in the periplasm and the other in the cytoplasm, connected by two hydrophobic transmembrane segments. The periplasmic domains contain recognition sites for attractant amino acids or for ligand-occupied conformations of binding proteins specific for attractant sugars or dipeptides (6, 11,19,20 temporal gradients created as it swims. The chemosensory system is designed to respond to changes in the environment, and thus cells exhibit sensory adaptation. The system adapts to changes in ligand occupancy by covalent modification of occupied transducer proteins at specific residues in the cytoplasmic domain (30). Four or five glutamates, depending upon the specific transducer (17, 21, 32), serve as methyl-accepting sites, becoming y-carboxyl methyl esters in a reaction catalyzed by CheR, a transducer-specific methylLransferase (31). The methyl esters are hydrolyzed by the CheB protein, ...

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A description of the efficient high-level expression of the monomer hemoglobin (GMG4) from Glycera dibranchiata is presented. The cDNA described by Simons and Satterlee [Simons, P.C., & Satterlee, J.D. (1989) Biochemistry 28, 8525-8530] was subcloned into an expression system, and conditions were found that led to the production of large amounts of soluble apoprotein (rec-gmg). These conditions included lowering the temperature during the induction period and growth in a rich medium with a higher ionic strength. Characterization of this reconstituted recombinant protein showed that it was not identical to the native GMH4 protein. Both UV-visible and 1H NMR data indicated differences within the holoprotein (rec-gmh) heme pocket compared to the native protein, the major difference being that two nonidentical heme orientations are significantly populated in rec-gmh. This phenomenon has been seen previously in other heme proteins, where these heme orientational isomers are described by a 180-deg rotation about the heme alpha-gamma meso axis. This work prompted the production of a complete chemical sequence for the native GMH4 [Alam S.L., Satterlee, J. D., & Edmonds, C. G. (1994) J. Protein Chem. 13, 151-164], which showed that the expressed rec-gmg protein differed at three primary sequence positions (41, 95, and 123) from the native component IV globin (GMG4). Subsequently, we have produced the triple-revertant mutations required to express the recombinant wild-type protein (recGMG4). The physical characteristics of the active site in the holoprotein (recGMH4) are identical to those of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)