Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance

Schuster, Stephan C.; Swanson, Ronald V.; Alex, Lisa A.; Bourret, Robert B.

doi:10.1038/365343a0

Cited by 255 publications

(195 citation statements)

References 24 publications

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“…In vitro protein-protein interaction experiments indicate that CheW and CheA from E. coli are capable of forming a thermodynamically stable complex with chemoreceptors under physiological conditions (11,35). If R. centenum CheWCheAY forms a stable complex with receptors, communication with the polar flagellar motor would require that receptors be located near flagella.…”

Section: Discussionmentioning

confidence: 99%

“…The construct was transformed in E. coli BL21 (DE3)/pLysS for inducible expression by isopropyl thiogalactopyranoside (IPTG; final concentration, 1.5 mM). Selective labeling of protein(s) expressed from the T7 promoter with 35 S was achieved by treating cells with rifampin prior to addition of [ 35 S]methionine as described previously (41,42). The CheAY polypeptide was visualized by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and standard autoradiography (33).…”

Section: Methodsmentioning

confidence: 99%

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Analysis of a chemotaxis operon from Rhodospirillum centenum

Jiang

Bauer

1997

J Bacteriol

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A chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum has been cloned, sequenced, and analyzed for the control of transcription during swimmer-to-swarm cell differentiation. The first gene of the operon (cheAY) codes for a large 108-kDa polypeptide with an amino-terminal domain that is homologous to CheA and a carboxyl terminus that is homologous to CheY. cheAY is followed by cheW, an additional homolog of cheY, cheB, and cheR. Sequence analysis indicated that all of the che genes are tightly compacted with the same transcriptional polarity, suggesting that they are organized in an operon. Cotranscription of the che genes was confirmed by demonstrating through Western blot analysis that insertion of a polar spectinomycin resistance gene in cheAY results in loss of cheR expression. The promoter for the che operon was mapped by primer extension analysis as well as by the construction of promoter reporter plasmids that include several deletion intervals. This analysis indicated that the R. centenum che operon utilizes two promoters; one exhibits a 70 -like sequence motif, and the other exhibits a 54 -like motif. Expression of the che operon is shown to be relatively constant for swimmer cells which contain a single flagellum and for swarm cells that contain multiple lateral flagella.More than a century ago, Engelmann (7) observed that when purple sulfur photosynthetic bacteria of the genus Chromatium were illuminated with a broad spectrum of light, the cells accumulated at wavelengths that corresponded to the in vivo absorption peaks of the cells. A more accurate description of this phenomenon is that when smooth-swimming cells migrate into a dark region (or into regions of the spectrum where wavelengths are not absorbed by photopigments), they either tumble or reverse the direction of movement, depending on the species. The phenomenon has been termed a scotophobic (fear of darkness) response since the cells exhibit an aversion to darkness rather than a specific affinity for light (12,29). Since a reduction in light intensity causes a tumbling/reversal response, the mechanism of moving through an increasing gradient of light intensity appears to involve a directed "random walk" such that the length of smooth swimming is longer when cells are going up a light gradient than when they are going down. Superficially, this process is not unlike the wellcharacterized bacterial "trial-and-error" walking up a chemical gradient that is mediated by chemoreceptors and the Che protein phosphorylation cascade (2, 40).The mechanism whereby a reduction in light intensity causes a tumbling/reversal response is still unclear. However, recent work has revealed that a functioning photosynthetic apparatus is required for photosensory perception (3). In addition, chemical inhibition of electron carrier components such as the cytochrome bc 1 complex, are incapable of light perception (4). These results have led to the current dogma that the scotophobic response is mediated by the perception of a sudden decre...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Analysis of a chemotaxis operon from Rhodospirillum centenum

Jiang

Bauer

1997

J Bacteriol

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“…On the other hand, the affinity-purified cyclin G, after removal of the GST portion by treatment with thrombin, displayed a single band at the expected size on SDS-PAGE (data not shown). Analysis by surface plasmon resonance biosensor technology [11,[27][28][29] suggested that GAK directly associates with GST-cyclin G, probably via the kinase domain at the Nterminus of the molecule. In this experiment, anti-GST antibody was covalently attached to the dextran matrix of a BIAcore sensor chip to assess the association at a neutral pH value.…”

Section: Analysis Of the Cyclin G-gak Interaction Using Thementioning

confidence: 99%

GAK: a cyclin G associated kinase contains a tensin/auxilin‐like domain¹

Kanaoka

Kimura

Okazaki³

et al. 1997

FEBS Letters

123

166

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We have cloned a cDNA encoding a novel association partner of cyclin G by West-Western blotting. The cDNA encodes a protein that harbors a Ser/Thr protein kinase-like catalytic domain at the N-terminal. Hence, we named it GAK (cyclin G-associated kinase). The long C-terminal extension shares homology with tensin and auxilin, and contains a leucine zipper region. Co-immunoprecipitation and Western blotting showed that GAK and cyclin G associate together in vivo. GAK also co-precipitated with CDK5, and CDK5 was found to be associated with cyclin G. We also showed by BIAcore analysis that the GAK-cyclin G interaction was direct.

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“…This possibility is consistent with results from 1 H-and 15 N-NMR spectroscopy on CheY D13K, which allow for a small percent of the D13K mutant molecule to be in an alternate conformation, rapidly interconverting with its resting state. 2 Comparison of Wild-type and CheY D13K Activities-CheY binds specifically to three different proteins: CheA, which donates the phosphoryl group to CheY (43)(44)(45)(46); CheZ, which stimulates dephosphorylation of CheY-P (43,(47)(48)(49); and FliM, the recipient of the activating signal of CheY, located in the flagellar switch complex (34,50). Phosphorylation of wild-type CheY reduces binding to CheA but enhances binding to CheZ and FliM.…”

Section: å X-ray Structure Of Chey Mutant D13kmentioning

confidence: 99%

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

Meiying

Bourret

Volz

1997

Journal of Biological Chemistry

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An aspartate to lysine mutation at position 13 of the chemotaxis regulatory protein CheY causes a constitutive tumbly phenotype when expressed at high copy number in vivo even though the mutant protein is not phosphorylatable. These properties suggest that the D13K mutant adopts the active, signaling conformation of CheY independent of phosphorylation, so knowledge of its structure could explain the activation mechanism of CheY. The x-ray crystallographic structure of the CheY D13K mutant has been solved and refined at 2.3 Å resolution to an R-factor of 14.3%. The mutant molecule shows no significant differences in backbone conformation when compared with the wild-type, Mg 2؉ -free structure, but there are localized changes within the active site. The side chain of lysine 13 blocks access to the active site, whereas its ⑀-amino group has no bonding interactions with other groups in the region. Also in the active site, the bond between lysine 109 and aspartate 57 is weakened, and the solvent structure is perturbed. Although the D13K mutant has the inactive conformation in the crystalline form, rearrangements in the active site appear to weaken the overall structure of that region, potentially creating a metastable state of the molecule. If a conformational change is required for signaling by CheY D13K, then it most likely proceeds dynamically, in solution.Cells continuously monitor various chemical and physical parameters in their surrounding environment and use this information to implement appropriate adaptive responses to changing conditions. This vital task is accomplished by a cascade of transient protein phosphorylation and dephosphorylation events arranged so that the flow of phosphoryl groups reflects environmental conditions. One ubiquitous class of signal transduction networks is the "two-component" regulatory systems that control diverse processes such as behavior, development, physiology, and virulence in bacteria, as well as adaptive processes in eukaryotes (for reviews, see Refs.

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Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance

Cited by 255 publications

References 24 publications

Analysis of a chemotaxis operon from Rhodospirillum centenum

Analysis of a chemotaxis operon from Rhodospirillum centenum

GAK: a cyclin G associated kinase contains a tensin/auxilin‐like domain¹

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

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Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance

Cited by 255 publications

References 24 publications

Analysis of a chemotaxis operon from Rhodospirillum centenum

Analysis of a chemotaxis operon from Rhodospirillum centenum

GAK: a cyclin G associated kinase contains a tensin/auxilin‐like domain1

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

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GAK: a cyclin G associated kinase contains a tensin/auxilin‐like domain¹