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R C Stewart

Publications and source records attributed to R C Stewart.

At least 19 recordsLinked to original sources
PubMed1999

Kinetic characterization of CheY phosphorylation reactions: comparison of P-CheA and small-molecule phosphodonors.

In the chemotaxis system of Escherichia coli, phosphorylation of the CheY protein plays an important role in regulating the swimming pattern of the cell. In vitro, CheY can be phosphorylated either by phosphotransfer from phospho-CheA or by acquiring a phosphoryl group from any of a variety of small, high-energy phosphodonor molecules such as acetyl phosphate. Previous work explored the rapid kinetics of CheY phosphorylation by CheA. Here we extend that work and examine the kinetics of CheY phosphorylation by several small-molecule phosphodonors, including acetyl phosphate, benzoyl phosphate, carbamoyl phosphate, 2-methoxybenzoyl phosphate, and phosphoramidate. Our results indicate that these phosphodonors bind to CheY with relatively low affinity (Ks values ranging from 10 to 600 mM) and that the rate constant (kphos) for phosphotransfer at saturating phosphodonor concentrations is relatively slow (values ranging from 0.05 to 0.5 s-1). By contrast, under identical conditions, phosphorylation of CheY by phospho-CheA occurs much more rapidly (kphos approximately 800 s-1) and reflects CheY binding to phospho-CheA considerably more tightly (Ks approximately 60 microM) than it does to the small-molecule phosphodonors. In comparing CheA-mediated phosphorylation of CheY to small-molecule-mediated phosphorylation of CheY, the large difference in kphos values suggests that phospho-CheA makes significant contributions to the catalysis of CheY phosphorylation. The effects of pH and ionic strength on CheY phosphorylation kinetics were also investigated. For CheA-->CheY phosphotransfer, increasing ionic strength resulted in increased Ks values while kphos was unaffected. For CheY phosphorylation by small-molecule phosphodonors, increasing ionic strength resulted in decreasing Ks values and increasing kphos values. The significance of these effects is discussed in relation to the catalytic mechanism of CheY phosphorylation by phospho-CheA and small-molecule phosphodonors.

Bacterial Proteins↗
PubMed1998

TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli.

The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli. This interaction results in autophosphorylation of CheA, a histidine protein kinase. Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors. As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals. To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides. Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently. Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP. Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP. Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner. TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM). The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made. Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase. This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA. These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA.

Adenosine Diphosphate↗
PubMed1998

Yeast Skn7p activity is modulated by the Sln1p-Ypd1p osmosensor and contributes to regulation of the HOG pathway.

Activation and control of the yeast HOG (High Osmolarity Glycerol) MAP kinase cascade is accomplished, in part, by a two-component sensory-response circuit comprised of the osmosensing histidine protein kinase Sln1p, the phospho-relay protein Ypd1p, and the response regulator protein Ssk1p. We found that deletion of SLN1 and/or YPD1 reduces reporter gene transcription driven by a second two-component response regulator -- Skn7p. The effect of sln1delta and ypd1delta mutations upon Skn7p activity is dependent on a functional two-component phosphorylation site (D427) in Skn7p, suggesting that Sln1p and Ypd1p may act as phosphodonors for Skn7p. We also observed that loss of PTC1 (a protein serine/threonine phosphatase implicated in negative control of the HOG pathway) in a skn7delta background results in severely retarded growth and in morphological defects. Deletion of either PBS2 or HOG1 alleviates the slow growth phenotype of ptc1delta skn7delta cells, suggesting that Skn7p may participate, in concert with known regulatory components, in modulating HOG pathway activity. The contribution of Skn7p to HOG pathway regulation appears to be modulated by the receiver domain, since non-phosphorylatable Skn7pD427N is unable to fully restore growth to ptc1/skn7 cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Kinetic characterization of phosphotransfer between CheA and CheY in the bacterial chemotaxis signal transduction pathway.

Phosphorylation of the CheY protein is a crucial step in the chemotaxis signal transduction pathway of Escherichia coli. CheY becomes phosphorylated by acquiring a phosphoryl group from CheA, an autophosphorylating protein kinase. In this study, we utilized a rapid-quench instrument to investigate the kinetics of phosphotransfer in single-turnover experiments. Our results are consistent with a three-step mechanism for the CheA-to-CheY phosphotransfer reaction: (i) reversible binding of CheY to P-CheA; (ii) rapid, reversible phosphotransfer to CheY; (iii) reversible dissociation of the resulting CheA x CheY-P complex. Investigation of the effect of CheY concentration on the observed rate of phosphotransfer demonstrated saturation kinetics; the extrapolated limiting rate constant for phosphotransfer was 650 +/- 200 s(-1), while the Km value indicated from this work was 6.5 +/- 2 microM. We demonstrated that the CheA-CheY phosphotransfer reaction was reversible by observing partial transfer of [32P]phosphate from CheY-P to CheA and by observing the effect of high concentrations of unphosphorylated CheA on the equilibrium: P-CheA + CheY <--> CheA + CheY-P. We found that the rate of phosphotransfer from P-CheA to CheY can be inhibited by unphosphorylated CheA as well as by a fragment of CheA (CheA124-257) that contains the CheY binding site; these results suggest that the unphosphorylated form of CheA can effectively compete with P-CheA for available CheY (Kd approximately 1.5 +/- 0.6 microM for the CheY x CheA124-257 complex and for the CheY x CheA complex).

Bacterial Proteins↗
PubMed1996

Identification of ASK10 as a multicopy activator of Skn7p-dependent transcription of a HIS3 reporter gene.

Recent evidence has demonstrated that the yeast Skn7p appears to act as a 'response regulator' in a eukaryotic 'two-component' signal transduction pathway. A search to identify possible regulators of the SKN7 mediated 'two-component' regulatory system has identified Ask10p as a novel potential transcription factor. The ASK10 sequence has been deposited in GenBank with Accession Number U27209.

Amino Acid Sequence↗
PubMed1994

Yeast Skn7p functions in a eukaryotic two-component regulatory pathway.

Previous analysis of the amino acid sequence of Skn7p, the product of the yeast SKN7 gene, revealed a potential 'receiver motif' homologous to that found in bacterial response regulators (signal-transducing effector proteins regulated by phosphorylation at a conserved aspartate residue corresponding to position D427 in Skn7p). We determined the effects of D427N and D427E mutations in Skn7p. The D427N substitution resulted in diminished activity in four independent in vivo assays of Skn7p function, while the D427E mutation enhanced Skn7p activity in these assays. Our results are consistent with predictions based on the bacterial two-component paradigm and provide experimental evidence that a receiver motif functions in regulating the activity of Skn7p in a eukaryote. Skn7p suppressed growth defects associated with a pkc1 delta mutation, raising the possibility that PKC1 might play a role in regulating Skn7p. However, epistasis experiments indicate that Skn7p does not appear to function directly downstream of the PKC1-MAP kinase pathway. Rather, Skn7p may function in a two-component signal transduction pathway that acts in parallel with the PKC1 cascade to regulate growth at the cell surface. We present evidence suggesting that Skn7p serves as a transcription factor in such a signaling pathway.

Amino Acid Sequence↗
PubMed1994

Kinetics of CheA autophosphorylation and dephosphorylation reactions.

The protein kinase CheA of Escherichia coli plays a central role in the signal transduction pathway controlling the swimming behavior of the cell in response to extracellular chemical gradients. CheA autophosphorylates at a rate controlled by the ligand binding state of chemotaxis receptor/transducer proteins. CheA directs the activities of CheY and CheB, effector proteins that become phosphorylated as a result of their interaction with phospho-CheA. In this study, we performed a detailed kinetic analysis of CheA's autophosphorylation reaction, and its dephosphorylation by ADP. Our kinetic data are consistent with a three-step mechanism for CheA autophosphorylation/dephosphorylation involving (i) substrate binding, (ii) phospho-transfer, and (iii) product release. We determined the dissociation constant for the kinetically defined CheA.ATP complex to be approximately 300 microM and the limiting rate constant for autophosphorylation to be approximately 0.026 s-1 at saturating ATP concentration. Our results indicate that the apparent dissociation constant of the phospho-CheA.ADP complex is approximately 42 microM and that the limiting rate constant for CheA dephosphorylation is approximately 0.028 s-1 at saturating ADP concentration. We corroborated the kinetically determined Kd values by performing independent ligand binding experiments. In addition, we found that the kinetics of trans-phosphorylation, involving mutant proteins CheA48HQ and CheA470GK, exhibited kinetic properties similar to those observed for autophosphorylation of wild-type CheA, although the limiting rate constant (0.008 s-1) was somewhat slower for this trans-phosphorylation reaction. These results will provide a framework for assessing the effects of various cheA mutations as well as for exploring the nature of CheA regulation by the chemotaxis receptor/transducer proteins.

Adenosine Diphosphate↗
PubMed1994

Pore geometry information via pulsed field gradient NMR.

Studies of echo attenuation at long diffusion times in pulsed field gradient NMR experiments on a variety of rock core samples are interpreted in the light of recent theoretical analysis of the effect of pore geometry and surface relaxation. This study is motivated by the need to test the applicability of that theory to real rock systems.

Calcium Carbonate↗
PubMed1994

3D autocorrelation for the determination of large pore sizes.

A data analysis methodology is used to process 3D NMR image data acquired for porous systems. The method extracts the mean size of those repeating elements in the image data which are largely compared with the image voxel dimensions. In this work we extend the two-dimensional (2D) image analysis method described by others to three spatial dimensions (3D). 3D image data were acquired at a magnetic field strength of 7 T using NMR microscopy hardware. The 3D autocorrelation function obtained from the data reveals a characteristic pore size in each dimension.

Image Processing, Computer-Assisted↗
PubMed1994

Strategies for overcoming linewidth limitations in quantitative petrophysical NMR measurements.

The simple pulse-acquire experiment has been used to evaluate the level of accuracy and precision achievable in NMR fluid saturation measurements for a range of rock core samples saturated with either brine or hydrocarbons. For a set of more than 70 cores measured at 0.66 T the mean error in the NMR measurement is only 0.35% porosity when the sample linewidths are less than 50 ppm. However, for a significant portion of cores, those with very broad NMR linewidths (> 50 ppm), difficulties associated with nonuniform excitation are encountered. The magnetic susceptibility difference between pore fluid and rock matrix translates into relatively broad NMR linewidths, and this feature of petrophysical samples is the major difficulty in performing quantitative NMR experiments. Numerical simulations are used to complement the experimental results in order to develop strategies for obtaining accurate NMR results with these difficult samples.

Hydrocarbons↗
PubMed1994

Quantitative longitudinal fluid saturation profiles with a slice-selected CPMG sequence.

A technique for obtaining quantitative longitudinal saturation/porosity profiles of rock cores which are longer than the NMR coil has been developed. The slice-selected experiment uses a prefocused pulse in conjunction with a magnetic field gradient for the localization and a CPMG sequence to sample the data. A variety of rock core samples has been studied ranging from limestones to shaly sandstones. Comparison of the relaxation decay curves obtained from these experiments and the bulk experiments show that reliable localized relaxation data are obtained.

Calcium Carbonate↗
PubMed1994

pH dependence of CheA autophosphorylation in Escherichia coli.

Chemotaxis by cells of Escherichia coli and Salmonella typhimurium depends upon the ability of chemoreceptors called transducers to communicate with switch components of flagellar motors to modulate swimming behavior. This communication requires an excitatory pathway composed of the cytoplasmic signal transduction proteins, CheAL, CheAS, CheW, CheY, and CheZ. Of these, the autokinase CheAL is most central. Modifications or mutations that affect the rate at which CheAL autophosphorylates result in profound chemotactic defects. Here we demonstrate that pH can affect CheAL autokinase activity in vitro. This activity exhibits a bell-shaped dependence upon pH within the range 6.5 to 10.0, consistent with the notion that two proton dissociation events affect CheAL autophosphorylation kinetics: one characterized by a pKa of about 8.1 and another exhibiting a pKa of about 8.9. These in vitro results predict a decrease in the rate of CheAL autophosphorylation in response to a reduction in intracellular pH, a decrease that should cause increased counterclockwise flagellar rotation. We observed such a response in vivo for cells containing a partially reconstituted chemotaxis system. Benzoate (10 mM, pH 7.0), a weak acid that when undissociated readily traverses the cytoplasmic membrane, causes a reduction of cytoplasmic pH from 7.6 to 7.3. In response to this reduction, cells expressing CheAL, CheAS, and CheY, but not transducers, exhibited a small but reproducible increase in the fraction of time that they spun their flagellar motors counterclockwise. The added presence of CheW and the transducers Tar and Trg resulted in a more dramatic response. The significance of our in vitro results, their relationships to regulation of swimming behavior, and the mechanisms by which transducers might affect the pH dependence of CheA autokinase activity are discussed.

Adenosine Triphosphate↗
PubMed1994

Mutational activation of CheA, the protein kinase in the chemotaxis system of Escherichia coli.

In Escherichia coli and Salmonella typhimurium, appropriate changes of cell swimming patterns are mediated by CheA, an autophosphorylating histidine protein kinase whose activity is regulated by receptor/transducer proteins. The molecular mechanism underlying this regulation remains unelucidated but may involve CheA shifting between high-activity and low-activity conformations. We devised an in vivo screen to search for potential hyperkinase variants of CheA and used this screen to identify two cheA point mutations that cause the CheA protein to have elevated autokinase activity. Each point mutation resulted in alteration of proline 337. In vitro, CheA337PL and CheA337PS autophosphorylated significantly more rapidly than did wild-type CheA. This rate enhancement reflected the higher affinities of the mutant proteins for ATP and an increased rate constant for acquisition by CheA of the gamma-phosphoryl group of ATP within a kinetically defined CheA.ATP complex. In addition, the mutant proteins reacted with ADP more rapidly than did wild-type CheA. We considered the possibility that the mutations served to lock CheA into an activated signaling conformation; however, we found that both mutant proteins were regulated in a normal fashion by the transducer Tsr in the presence of CheW. We exploited the activated properties of one of these mutants to investigate whether the CheA subunits within a CheA dimer make equivalent contributions to the mechanism of trans phosphorylation. Our results indicate that CheA trans phosphorylation may involve active-site residues that are located both in cis and in trans to the autophosphorylation site and that the two protomers of a CheA dimer make nonequivalent contributions in determining the affinity of the ATP-binding site(s) of CheA.

Adenosine Diphosphate↗
PubMed1994

The short form of CheA couples chemoreception to CheA phosphorylation.

Escherichia coli cells express two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in chemotactic signal transduction. As a histidine protein kinase, it first autophosphorylates at amino acid His-48; then, it phosphorylates two other chemotaxis proteins, CheY and CheB. The short form, CheAS, lacks the amino-terminal 97 amino acids of CheAL and, therefore, does not contain the site of autophosphorylation. However, it does retain a functional kinase domain. As a consequence, CheAS can mediate transphosphorylation of kinase-deficient CheAL variants. Here we demonstrate in vitro that CheAS also can mediate transphosphorylation of a CheAL variant that lacks the C-terminal segment, a portion of the protein which is thought to interact with CheW and the chemoreceptors. The presence of CheW and the chemoreceptor Tsr enhances this activity and results in modulation of the transphosphorylation rate in response to the Tsr ligand, L-serine. Because CheAS can mediate this activity, it can restore chemotactic ability to Escherichia coli cells that express this truncated CheAL variant.

Alleles↗
PubMed1993

CheZ mutants with enhanced ability to dephosphorylate CheY, the response regulator in bacterial chemotaxis.

CheZ is a component of the chemotaxis signal-transduction pathway in Escherichia coli and Salmonella typhimurium. It is responsible for accelerating dephosphorylation of CheY and thereby antagonizing the tumble-promoting activity of CheY. In the absence of functional CheZ, cells are non-chemotactic and tumble constantly. We characterized the effects of two mutations in CheZ, R54C and V166G, that are unusual in that they cause cells to have a smooth swimming bias. These mutations were isolated as second-site suppressors of mutations in the switch complex responsible for regulating the direction of flagellar rotation (Yamaguchi, S., Aizawa, S.-I., Kihara, M. Isomura, M., Jones, C.J. and Macnab, R.M. (1986) J. Bacteriol. 168, 1172-1179). When produced at low levels in a delta cheZ host strain, CheZ R54C and CheZ V166G supported chemotaxis. However, when moderately overproduced they markedly inhibited chemotactic ability. In vitro studies revealed that these mutations enhanced the ability of CheZ to accelerate dephosphorylation of CheY. These results are discussed in relation to the possible roles and interactions of CheZ in the chemotaxis system.

Alleles↗
PubMed1993

The short form of the CheA protein restores kinase activity and chemotactic ability to kinase-deficient mutants.

Escherichia coli expresses two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct, in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in the chemotactic signal transduction mechanism by phosphorylating two other chemotaxis proteins: CheY and CheB. CheAL must first autophosphorylate at amino acid His-48 before transferring its phosphono group to these other signal transduction proteins. The short form, CheAS, lacks the N-terminal 97 amino acids of CheAL and, therefore, does not possess the site of autophosphorylation. Here we demonstrate that although it lacks the ability to autophosphorylate, CheAS can mediate phosphorylation of kinase-deficient variants of CheAL each of which retains a functional autophosphorylation site. This transphosphorylation enables these kinase-deficient CheAL variants to phosphorylate CheY. Because it mediates this activity, CheAS can restore to kinase-deficient E. coli cells the ability to tumble and, thus, to perform chemotaxis in swarm plate assays.

Bacterial Proteins↗
PubMed1993

Activating and inhibitory mutations in the regulatory domain of CheB, the methylesterase in bacterial chemotaxis.

In the chemotaxis system of Escherichia coli, CheB promotes sensory adaptation by interacting with the chemotaxis receptor-transducer proteins to catalyze removal of their gamma-glutamyl methyl ester groups. CheB is comprised of two functional domains; the C-terminal domain contains the methylesterase active site, and the N-terminal domain regulates the activity of this active site. The chemotaxis system controls CheB methylesterase activity via a mechanism involving phosphorylation of the CheB regulatory domain by the chemotaxis protein kinase CheA. To further explore the communication between the regulatory and methylesterase domains of CheB, I generated mutations in the CheB regulatory domain that affect methylesterase activity in vitro. Three of these mutations (D11K, E58K, and E91K) caused increased methylesterase activity in the absence of phosphorylation, and several other mutations (R42H, R73H, and K107R) caused decreased methylesterase activity in the purified proteins. Several of these mutations (D10N, D11K, R42H, E58K, and K107R) also affected the phosphorylation biochemistry of CheB by reducing the rate of CheA-mediated phosphorylation of CheB and/or by decreasing the autodephosphorylation rate of CheB. In addition, all of these mutations diminished the ability of excess CheA to inhibit CheB methylesterase activity. The locations of these mutations in the deduced three-dimensional structure of the CheB N-terminal domain indicate that the region of the protein surrounding the putative phosphorylation site plays important roles in its interaction with the CheB C-terminal domain as well as in its interactions with CheA.

Adenosine Triphosphatases↗