RESUMEN
Transmembrane signaling by bacterial chemoreceptors is thought to involve conformational changes within a stable homodimer. We investigated the functional consequences of constraining movement between pairs of helices in the four-helix structure of the transmembrane domain of chemoreceptor Trg. Using a family of cysteine-containing receptors, we identified oxidation treatments for intact cells that catalyzed essentially complete sulfhydryl cross-linking at selected positions and yet left flagellar and sensory functions largely unperturbed. Constraining movement by cross-links between subunits had little effect on tactic response, but constraining movement between transmembrane segments of the monomer drastically reduced function. We deduce that transmembrane signaling requires substantial movement between transmembrane helices of a monomer but not between interacting helices across the interface between subunits.
Asunto(s)
Proteínas Bacterianas/fisiología , Quimiotaxis/fisiología , Proteínas de Escherichia coli , Escherichia coli/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Proteínas Bacterianas/química , Reactivos de Enlaces Cruzados , Relación Dosis-Respuesta a Droga , Escherichia coli/efectos de los fármacos , Flagelos/fisiología , Proteínas de la Membrana , Modelos Moleculares , Movimiento , Oxidación-Reducción , Conformación Proteica , Receptores de Superficie Celular/química , Ribosa/farmacología , Compuestos de SulfhidriloRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Células Quimiorreceptoras/química , Reactivos de Enlaces Cruzados/química , Disulfuros/química , Proteínas de Escherichia coli , Proteínas Bacterianas/genética , Membrana Celular/química , Proteínas de la Membrana , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Conformación ProteicaRESUMEN
halobacterium salinarium (formerly H. halobium) is a chemotactic and phototactic archaeon from which volatile methyl groups are released continually, a phenomenon related to its sensory system. We found that released methyl groups comprised two different chemical species, methanol and methanethiol, the sulfur analog of methanol. Radiolabeling experiments showed that the methyl groups of both compounds, as well as the sulfur of methanethiol, were derived from methionine but were donated to cellular components and subsequently cleaved to produce the respective volatile compounds. Previous work had shown that chemostimuli and photostimuli result in transient increases in the rate of release of volatile methyl groups. We found that these increases reflected increased release of methanol but not of methanethiol. Thus, the methyl group chemistry of the H. salinarium sensory system is analogous to the well-studied chemotactic system of Escherichia coli. The reactions that result in methanethiol release are of unknown function and have unusual features. They may involve a methionine-gamma-lyase activity we detected in H. salinarium. Sulfur derived from methionine was found attached to specific proteins in reduction-sensitive disulfide linkages.