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F W Dahlquist

Publications and source records attributed to F W Dahlquist.

At least 91 records · Page 5Linked to original sources
PubMed1985

Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme.

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.

Amides↗
PubMed1985

Aberrant regulation of methylesterase activity in cheD chemotaxis mutants of Escherichia coli.

The adaptation process in several cheD chemotaxis mutants, which carry defects in tsr, the serine transducer gene, was examined. cheD mutants are smooth swimming and generally nonchemotactic; the defect is dominant to the wild-type tsr gene (J. S. Parkinson, J. Bacteriol. 142:953-961, 1980). All classes of methyl-accepting chemotaxis proteins synthesized in unstimulated cheD strains are overmethylated relative to the wild type. We found that the steady-state rate of demethylation in cheD mutants was low; this may explain their overmethylated phenotype. In addition, all cheD mutants showed diminished responsiveness of methylesterase activity to attractant and repellent stimuli transduced by either the Tsr or Tar protein, and they did not adapt. These results suggest that the dominant nature of the cheD mutations is manifested as a general defect in the regulation of demethylation. Some of these altered properties of methylesterase activity in cheD mutants were exhibited in wild-type cells that were treated with saturating concentrations of serine. The mutant Tsr protein thus seems to be locked into a signaling mode that suppresses tumbling and inhibits methylesterase activity in a global fashion. We found that the Tar and mutant Tsr proteins synthesized in cheD strains were methylated and deamidated at the correct sites and that the mutations were not located in the methylated peptides. Thus, the signaling properties of the transducers may be controlled at sites distinct from the methyl-accepting sites.

Bacterial Proteins↗
PubMed1985

Sensory adaptation in bacterial chemotaxis: regulation of demethylation.

The behavioral responses of chemotactic bacteria to environmental stimuli are initiated by a family of membrane-bound transducer proteins that communicate excitatory signals to the flagellar apparatus. The adaptation process appears to turn off the excitatory signal and is mediated by the reversible methylation of multiple sites on the transducer proteins. The activities of two chemotaxis-specific enzymes, a methyltransferase and a methylesterase, are regulated during adaptation to maintain behavioral responsiveness. To monitor stimulus-induced changes in methylesterase activity in intact cells, we quantitated the continuous generation of methanol, the end product of the demethylation reaction, in a flow device. In this paper we describe studies of the regulation of the demethylation process. Changes in methylesterase activity after the simultaneous addition of opposing stimuli through two different transducer classes suggest that the sensory information detected by these transducers was integrated and that this integrated signal controlled demethylation.

Acclimatization↗
PubMed1984

Stimulus-induced changes in methylesterase activity during chemotaxis in Escherichia coli.

Responses of Escherichia coli to chemical stimuli are mediated by a family of sensory transducer proteins. These transmembrane proteins detect stimuli and convey sensory information to the flagella. Behavioral adaptation to environmental changes is correlated with the reversible methylation of the transducer proteins on several (4 to 6) specific glutamic acid residues to form methyl esters. We have assayed the activity of the chemotaxis-specific methylesterase, the product of the cheB gene, by measuring the product of the demethylation reaction, methanol, using a modification of a previous method (Toews, M.L., Goy, M.F., Springer, M.S., and Adler, J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5544-5548; Terwilliger, T.C., Bogonez, E., Wang, E.A., and Koshland, D. E., Jr. (1983) J. Biol. Chem. 258, 9608-9611). In this study, we establish the use of a sensitive flow apparatus for measuring stimulus-induced changes in methylesterase activity of intact E. coli cells. Responses to positive and negative stimuli consist of transient decrease and increases in methylesterase activity, respectively. In addition, chase experiments demonstrated that all assayable methyl groups are nearly equivalent. The results are not consistent with the view that the methyl groups put on the transducer proteins as a result of a positive stimulus are the first to be removed when that stimulus is withdrawn. It seems more likely that recently added methyl groups become part of a pool of kinetically equivalent members, any of which can be removed when the stimulus is withdrawn. The flow measurements provide a powerful and simple biochemical assay that complements behavioral studies of chemotaxis.

Bacterial Proteins↗
PubMed1983

Multiple covalent modifications of Trg, a sensory transducer of Escherichia coli.

The sensory transducers of Escherichia coli are integral membrane proteins that mediate the tactic response of cells to chemical stimuli. Adaptation to environmental stimuli is correlated with methylation of the transducer proteins. Two transducer genes, tsr and tar, exhibit extensive homologies while no homology has been detected between a third transducer, trg, and those genes. The Tsr and Tar proteins have been shown to contain multiple sites for methylation as well as two sites for another modification that requires an active cheB gene product and is designated the CheB-dependent modification. In this study, covalent modifications of the Trg protein were characterized by analysis of tryptic peptides. We found that methylation occurred at several sites on the Trg protein and that the protein contained at least three sites for CheB-dependent modification, two of which were located on a tryptic peptide that contains both methionine and lysine. This tryptic peptide is analogous to the methionine- and lysine-containing methyl-accepting peptides isolated from the Tsr and Tar proteins and like those peptides may contain several methyl-accepting sites. We estimated the pKa of the group created by the CheB-dependent modification on the methionine- and lysine-containing peptide of Trg to be between pH 2.2 and 5.8. This result supports the idea that the CheB-dependent modification is an enzymatic deamidation of glutamine to glutamic acid.

Bacterial Proteins↗
PubMed1983

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins.

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.

Chromatography, High Pressure Liquid↗
PubMed1983

31P-NMR analysis of sea urchin sperm activation. Reversible formation of high energy phosphate compounds by changes in intracellular pH.

31P-NMR has been used to estimate the internal pH (pHi) of sperm from the sea urchin Strongylocentrotus purpuratus. The values for pHi obtained from the chemical shift of inorganic phosphate agree well with those obtained from amine accumulation. At low pHi, when sperm are quiescent (immotile and non-respiring), they accumulate phosphocreatine (PCr), but have a low level of inorganic phosphate (Pi). Conversely, when the pHi is elevated, sperm respiration and motility are activated, PCr is decreased and Pi is increased. This change is reversible upon decrease of the pHi, whereupon respiration and motility are arrested, Pi disappears and PCr increases. We conclude that the overall balance of energy metabolism, and thus the phosphate potential, of sea urchin sperm are under the control of the pHi.

Adenosine Triphosphate↗
PubMed1983

Enzymatic deamidation of methyl-accepting chemotaxis proteins in Escherichia coli catalyzed by the cheB gene product.

The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli undergo reversible methylation that has been correlated with adaptation of cells to environmental stimuli. MCPI, the product of the tsr gene, accepts methyl groups at multiple sites that are located on two tryptic peptides, denoted K1 and R1. A second modification of the MCPs, which is not methylation, has been designated the CheB-dependent modification. A CheB-dependent modification occurs on methyl-accepting peptide K1 and allows additional methyl groups to be incorporated into this peptide. We have performed partial amino acid sequence analyses on radiolabeled peptides K1 and R1 derived from MCPI and have identified several methyl-accepting sites. We found that, in the absence of CheB-dependent modification, a site in peptide K1 is unable to accept methyl groups. Correlation of this protein sequence data with the nucleotide sequence of the tsr gene [Boyd, A., Kendall, K. & Simon, M.I. (1983) Nature (London) 301, 623-626] suggests that CheB-dependent modification of MCPI is the enzymatic deamidation of glutamine to methyl-accepting glutamic acid. Possible roles for this deamidation in bacterial chemotaxis are discussed.

Amidohydrolases↗
PubMed1982

Fluorinated ligands as nuclear magnetic resonance probes of active-site nonequivalence in abortive ternary complexes of horse liver alcohol dehydrogenase.

The interactions of the dimeric horse liver alcohol dehydrogenase (LADH) with ligands in two nonreactive ternary complexes have been examined by using nuclear magnetic resonance and fluorescence techniques. One complex contains the enzyme, NADH, and the abortive alcohol substrate p-(trifluoromethyl) benzyl alcohol. The trifluoromethyl group allows the bound environment and bound to free exchange kinetics of this alcohol to be examined by using 19F NMR. Binding isotherms of the coenzyme and the abortive substrate were examined by using fluorescence. Similar measurements were made with the enzyme, NADH, and the aldehyde analogue p-(trifluoromethyl) benzamide. For both complexes there was no evidence of cooperative equilibrium binding of any ligand. Careful measurements of the exchange kinetics of the fluorinated alcohol or amide when binding to the enzyme NADH complex using NMR techniques showed that a single lifetime describes the exchange of ligands from both subunits of the protein. These results appear to rule out any site-site interactions in this system and support the notion that the observed biphasic kinetics observed in transient reactions of LADH with NAD and alcohols is a property of a single site in this system.

Alcohol Dehydrogenase↗
PubMed1982

The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I.

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.

Bacterial Proteins↗
PubMed1982

Halibut muscle 3-phosphoglycerate kinase. Chemical and physical properties of the enzyme and its substrate complexes.

An efficient procedure for the purification of 3-phosphoglycerate kinase (PGK) from Pacific halibut muscle is described. The molecular weight (43500) and specific activity are similar to those of other species of PGK. The isoelectric point (greater than 9.5) is more than 1.4 pH units higher than that reported for mammalian muscle PGK. The reaction of the seven thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is kinetically biphasic; reaction at a single fast-reacting thiol inactivates the enzyme. The binding of all substrates and products to PGK was observed by 31P NMR. 1,3-Diphosphoglycerate (1,3-P2G) is more tightly bound than is any of the other reaction components. Unlike 1,3-P2G in aqueous solution, the complex with PGK is protected from hydrolysis over a period of weeks. The 31P chemical shifts of this complex are insensitive to pH which suggests that solvent water is excluded from the substrate-bound cleft. As with yeast PGK, the equilibrium constant for the phosphoryl transfer reaction is near unity in the enzyme site environment in contrast to a value of approximately 10(3) (in favor of ATP) in aqueous solution. Since the ternary complex equilibrium 31P NMR spectrum can be accounted for entirely on the basis of the various binary complex spectra, there is no compelling evidence for the involvement of a stoichiometrically substantial phosphoenzyme intermediate.

Adenosine Triphosphate↗
PubMed1982

Properties of bound trifluoroethanol complexes with horse liver alcohol dehydrogenase.

The substrate analogue 2,2,2-trifluoroethanol (TFE) has been used as a 19F NMR probe of the active site of alcohol dehydrogenase from horse liver (LADH). We are able to directly observe a single resonance assigned to TFE in its ternary complex with LADH and nicotinamide adenine dinucleotide (NAD). The chemical shift of this resonance is independent of pH between values of 6.2 and 8.9, suggesting that bound TFE does not change ionization state in this range. Both by 19F NMR self-exchange measurements and by ligand-displacement studies with pyrazole, we also find that displacement of TFE from its ternary complex with NAD is a linear function of proton concentration over a similar pH range, with more rapid desorption occurring at lower pH values. This suggests that the pK of 6.4 for this process seen previously by Kvassman and Pettersson [Kvassman, J., & Pettersson, G. (1980) Eur. J. Biochem. 103, 557] is not due to the ionization of bound TFE. These studies also show that the bound lifetime of TFE in its ternary complex with LADH and NAD is quite long (400s) at pH 8.7, suggesting the use of TFE as a kinetic trapping reagent in single-turnover stopped-flow experiments. Binding isotherms of NAD to LADH saturated with TFE at pH 8.7 or with pyrazole at pH 7.5 reveal essentially no cooperative behavior. The displacement time courses described above are all adequately fit as first-order processes, thus giving no evidence for site heterogeneity or site-site interaction in the binding of these ligands to dimeric LADH. TFE and pyrazole are used as reagents to further explore the question of site-site interaction from a kinetic point of view in the following paper [Anderson, D. C., & Dahlquist, F.W. (1982) Biochemistry (following paper in this issue)].

Alcohol Dehydrogenase↗
PubMed1982

Biphasic transient kinetics are a property of a single site in horse liver alcohol dehydrogenase.

The transient kinetics under single turnover conditions of the dimeric enzyme horse liver alcohol dehydrogenase (LADH) are often biphasic with nearly equal amplitudes in the fast and slow components of the reaction. These observations have been the basis for the suggestion that this enzyme displays half of the sites reactivity or kinetic site-site interactions in its catalytic cycle [Bernhard, S.A., Dunn, M.F., Luisi, P.L., & Shack, P. (1970) Biochemistry 9, 185]. Alternatively, the biphasic kinetics could be the result of a complex mechanistic path at a single site with no kinetic interactions between the active sites [Kvassman, J., & Pettersson, G. (1976) Eur. J. Biochem. 62, 279; Kvassman, J., & Pettersson, G. (1978) Eur. J. Biochem. 87, 417]. To resolve this question, we have prepared LADH in which one of the two active sites is occupied by the strongly bound, slowly exchanging ternary complex inhibitors NAD with trifluoroethanol or NAD with pyrazole. These half-inhibited dimers show essentially the same biphasic transient kinetics as the native enzyme. These results strongly support mechanisms in which the observed kinetics are a property of each independently functioning site of the dimer. The possibility of half of the sites reactivity or kinetic site-site interaction appears to be ruled out.

Alcohol Dehydrogenase↗
PubMed1982

Adaptation in bacterial chemotaxis: CheB-dependent modification permits additional methylations of sensory transducer proteins.

Sensory transduction in E. coli consists of two phases, excitation and adaptation, both of which involve the methyl-accepting chemotaxis proteins (MCPs). These molecules relay transmembrane signals and are reversibly methylated during adaptation of E. coli to environmental stimuli. Each MCP contains multiple sites of methylation, and we identified six of these sites in MCPI. Recently, a second covalent modification of MCPs has been identified, which is not methylation. This modification, designated CheB-dependent modification, is stimulated by repellents and causes a net increase in the negative charge of MCPI and MCPII by one or two charges. We demonstrate that one CheB modification occurs on the methyl-accepting methionine-and lysine-containing tryptic peptide in MCPI and MCPII, and the second CheB modification is on an arginine-containing tryptic peptide. The CheB modification allows three additional methyl groups to be incorporated into the methyl-accepting methionine-lysine peptide, while not actually creating all of these methylation sites. The two CheB modifications occur sequentially. A possible mechanism by which CheB modification permits additional methylations and the role of CheB modification in bacterial chemotaxis are discussed.

Bacterial Proteins↗
PubMed1981

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus.

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1981

Dynamical and temperature-dependent effects of lipid-protein interactions. Application of deuterium nuclear magnetic resonance and electron paramagnetic resonance spectroscopy to the same reconstitutions of cytochrome c oxidase.

2H NMR and EPR spectra have been obtained as a function of temperature and protein concentration from the same samples of beef heart cytochrome c oxidase reconstituted into 1-(16,16,16-trideuteriopalmitoyl)-2-palmitoleoyl-sn-glycero-3-phosphocholine. At all temperatures, the EPR spectra show the characteristic "bound" and "free" components, while the 2H NMR spectra show only a narrow distribution of orientational order parameters. At temperatures near the phase transition of the pure lipid, the dependence of the 2H NMR average orientational order on protein concentration fits a two-stage model in which the phospholipid molecular exchange rapidly between two states tentatively identified as sites either on or off the protein surface. From this model, the 2H NMR spectra yield a value of 0.18 mg of phospholipid per mg of protein as necessary to cover the surface of cytochrome c oxidase, which is the same value as derived from the EPR spectra at -20 degrees C. Both the 2H NMR and EPR spectra vary markedly with temperature. At temperatures well above the phase transition of the pure lipid, the average orientational parameters derived from the 2H NMR spectra are independent of protein concentration and are the same as for the lipid alone. Qualitatively, the EPR spectra show large apparent decreases in the average orientational order with increasing temperature. Analysis of 2H NMR relaxation rates indicates an additional motion in the presence of protein with a correlation time of 10(-6)-10(-7) s. If this new motion is associated with exchange between the two states, a minimum value of 10(6)-10(7) s-1 for the exchange rate is obtained, assuming that the lipids on the protein surface are much more motionally restricted than the rest of the lipid. Such an exchange rate is compatible with the observed differences in 2H NMR and EPR spectra. These results are consistent with short-lived, energetically weak interactions between cytochrome c oxidase and the phospholipids used in this study.

Animals↗