RESUMEN
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.
Asunto(s)
Proteínas de la Membrana/genética , Mutagénesis , Membrana Celular/metabolismo , Quimiotaxis , Cisteína/análisis , Análisis Mutacional de ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Conformación Proteica , Transducción de SeñalRESUMEN
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
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)
Asunto(s)
Anélidos/genética , Hemoglobinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Hemo/química , Hemoglobinas/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , EspectrofotometríaRESUMEN
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(5E), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(5Q), 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 signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(5E) 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.