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Biomedical subjects

D Wemmer

Publications and source records attributed to D Wemmer.

At least 19 recordsLinked to original sources
PubMed2001

BeF(3)(-) acts as a phosphate analog in proteins phosphorylated on aspartate: structure of a BeF(3)(-) complex with phosphoserine phosphatase.

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF(3)(-), which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-A resolution the x-ray crystal structure of the complex of BeF(3)(-) with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF(3)(-) is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg(2+), and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF(3)(-) x PSP and BeF(3)(-) x CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF(3)(-) for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Aspartic Acid↗
PubMed1998

Reading DNA.

A new structure beings into sharp focus the structural basis for G-C base pair recognition by small ligands in the minor groove of DNA. At the same time the ability to distinguish specific sequences moves another major step forward through introduction of a new ligand modification.

Base Composition↗
PubMed1997

Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: evidence for an activating surface.

The bacterial enhancer-binding protein NtrC activates transcription when phosphorylated on aspartate 54 in its amino (N)-terminal regulatory domain or when altered by constitutively activating amino acid substitutions. The N-terminal domain of NtrC, which acts positively on the remainder of the protein, is homologous to a large family of signal transduction domains called receiver domains. Phosphorylation of an aspartate residue in a receiver domain modulates the function of a downstream target, but the accompanying structural changes are not clear. In the present work we examine structural and functional differences between the wild-type receiver domain of NtrC and mutant forms carrying constitutively activating substitutions. Combinations of such substitutions resulted in both increased structural changes in the N-terminal domain, monitored by NMR chemical shift differences, and increased transcriptional activation by the full-length protein. Structural changes caused by substitutions outside the active site (D86N and A89T) were not only local but extended over a substantial portion of the N-terminal domain including the region from alpha-helix 3 to beta-strand 5 ("3445 face") and propagating to the active site. Interestingly, the activating substitution of glutamate for aspartate at the site of phosphorylation (D54E) also triggered structural changes in the 3445 face. Thus, the active site and the 3445 face appear to interact. Implications with respect to how phosphorylation may affect the structure of receiver domains and how structural changes may be communicated to the remainder of NtrC are discussed.

Bacterial Proteins↗
PubMed1990

A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase.

An active site, cofactor-containing peptide has been obtained in high yield from bovine serum amine oxidase. Sequencing of this pentapeptide indicates: Leu-Asn-X-Asp-Tyr. Analysis of the peptide by mass spectrometry, ultraviolet-visible spectroscopy, and proton nuclear magnetic resonance leads to the identification of X as 6-hydroxydopa. This result indicates that, contrary to previous proposals, pyrroloquinoline quinone is not the active site cofactor in mammalian copper amine oxidases. Although 6-hydroxydopa has been implicated in neurotoxicity, the data presented suggest that this compound has a functional role at an enzyme active site.

Amine Oxidase (Copper-Containing)↗
PubMed1984

Mechanisms of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia.

We show that a transient lactic fermentation provides the signal triggering ethanol production in hypoxic maize root tips. The signal is cytoplasmic pH. This interaction between lactic and ethanolic fermentation permits tight cytoplasmic pH regulation during hypoxia--cytoplasmic pH remaining near neutrality for several hours. Mutant roots unable to synthesize ethanol can neither regulate cytoplasmic pH nor maintain ATP levels during extended periods of hypoxia and, like vertebrate tissues, are less tolerant of hypoxia than normal maize. This indicates that cytoplasmic pH regulation is an important factor in survival under hypoxia.

Cytoplasm↗
PubMed1984

Measurement of mitochondrial ATPase activity in maize root tips by saturation transfer p nuclear magnetic resonance.

We show that saturation transfer (31)P nuclear magnetic resonance can be used to measure the activity of the mitochondrial ATPase of maize (Zea mays L. hybrid WW x Br38) root tips in vivo. Unidirectional rates of ATP synthesis were determined in the steady state (i.e. ATP and cytoplasmic orthophosphate constant) under various conditions. These measurements, and determinations of oxygen consumption, give a P/O ratio (measured in the living tissue) close to 3. In succinate-fed root tips the P/O ratio is approximately 2. Cyanide inhibits the rate of ATP synthesis by two-thirds (P/O ratio approximately 1), with an effective inhibitor constant of approximately 35 micromolars. We show that the alternative electron pathway cannot make ATP, and does not normally operate in this tissue. This method of studying plant mitochondrial metabolism avoids potential artifacts encountered in studies using isolated and purified mitochondria. The method also allows, for the first time, direct and simultaneous examination of the relationship between the rate of ATP synthesis and levels of metabolites such as ATP, and derived parameters such as phosphorylation potential.

Journal Article↗
PubMed1983

Structure of apamin in solution: a two-dimensional nuclear magnetic resonance study.

A two-dimensional (2-D) Fourier-transform nuclear magnetic resonance (NMR) study of the 18 amino acid neurotoxin apamin isolated from honeybee venom is reported. Combining 2-D J-correlated spectra with 2-D nuclear Overhauser spectra in H2O solution permits essentially complete assignment of the 1H NMR spectrum of apamin at a fixed pH, including a number of spin systems that are reported for the first time. The structural model previously derived by Bystrov et al. [Bystrov, V. F., Okhanov, V. V., Miroshnikov, A. I., & Ovchinnikov, Yu. A. (1980) FEBS Lett. 119, 113-116] from NMR data is shown to be largely correct. The 2-D nuclear Overhauser effect (NOE) spectrum in particular reveals a series of amide-amide NOE's consistent with an alpha-helical core (residues 9-15) in the molecule. NOE's between amide and C alpha protons, followed by amide to amide NOE's, indicate a beta-turn involving residues 3-5 and a nonstandard turn including residues 6-8. We find no evidence for the beta-type structure postulated at the C terminus, however. Instead, it appears that the alpha-helix continues with increasing fraying from residues 16-18.

Apamin↗
PubMed1982

Continuous-flow NMR culture system for mammalian cells.

A continuous-flow NMR culture system for mammalian cells has been developed on which 31P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by 31P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a delta pH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.

Animals↗
PubMed1982

Regulation of Cytoplasmic and Vacuolar pH in Maize Root Tips under Different Experimental Conditions.

(31)P-Nuclear magnetic resonance spectra of perfused maize (Zea mays L., hybrid WW x Br 38) root tips, obtained at 10-minute intervals over 12 hours or longer, indicate that no cytoplasmic or vacuolar pH changes occur in these cells in the presence of 25 millimolar K(2)SO(4), which induces extrusion of 4 to 5 microequivalents H(+) per gram per hour. In contrast, hypoxia causes cytoplasmic acidification (0.3-0.6 pH unit) without a detectable change in vacuolar pH. The cytoplasm quickly returns to its original pH on reoxygenation. Dilute NH(4)OH increases the vacuolar pH more than it does the cytoplasmic pH; after NH(4)OH is removed, the vacuole recovers its original pH more slowly than does the cytoplasm. The results indicate that regulation of cytoplasmic pH and that of vacuolar pH in plant cells are separate processes.

Journal Article↗
PubMed1981

pH dependence of the high-resolution proton nuclear magnetic resonance spectrum of the lac repressor headpiece.

The pH dependence of the N-terminal 51 amino acid headpiece (HP) of the lac repressor has been followed by using 1H NMR spectroscopy to monitor the chemical shifts of resolved aromatic and methyl resonances. The NMR evidence reveals the folded HP domain to be stable from pH 1 to 10 at 23 degrees C. All observed resonances shift toward their expected random-coil positions above pH 10, which suggests that a general unfolding occurs. The four tyrosine rings reflect a combination of unfolding and titration in the order 7 greater than 17, 12 greater than 47. This pH-induced unfolding is completely reversible. In addition, strikingly similar pH behavior for selected tyrosine and methyl resonances at acid pH values suggests that clusters of various tyrosine, methyl, and carboxyl side chains exist in the native structure.

Amino Acids↗
PubMed1981

High-resolution proton nuclear magnetic resonance studies of the exchangeable resonances of the lac repressor headpiece.

The exchangeable hydrogens of the N-terminal 51 amino acid headpiece (HP) of the lac repressor have been examined with high-resolution 1H NMR. Resonances from 35 +/- 3 protons are observable in 2H2O solutions at p2H 4.2. The exchange rates for some of these have been measured over the p2H range 1.5-5. In H2O, resonances from 70 +/- 6 protons are observable at pH 3.2 and 35 +/- 5 protons at pH 7.1. The temperature dependence of the spectrum at neat neutral pH shows the existence of a group of approximately 22 protons particularly slow to exchange. The pH dependence of the spectrum in H2O solution over the range 1-11 is discussed. Results are compared with NMR studies of the nonexchanging protons and CD and IR results.

Deuterium↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 1. Assignments of tyrosine resonances in the N-terminal headpiece.

The DNA binding site of the lac repressor protein has been implicated to lie within the N-terminal 51 amino acid fragment termed headpiece (HP-51 or LR-51). High-resolution NMR suggests that isolated HP-51 retains most of the secondary and tertiary structure which it has in the whole repressor. Four of the eight tyrosines of repressor are in HP-51. 1H NMR spectra (360 MHz) over the aromatic region of native HP-51 show that the four tyrosines are nonequivalent with an unusual distribution of chemical shifts. Denaturation leads to loss of these chemical shift differences. Homonuclear decoupling and a two-dimensional autocorrelated spectrum allow unequivocal pairing of resonances from Tyr A at 6.99 and 6.79 ppm, Tyr B at 6.98 and 6.39 ppm, Tyr C at 6.70 and 6.54 ppm, and Tyr D at 6.39 and 6.33 ppm. The 2,6 protons are low field of the 3,5 protons for each Tyr residue. Selective chemical modification with nitration reagents allows assignments of Tyr A to Tyr-47, Tyr B to Tyr-7, Tyr C to Tyr-12, and Tyr D to Tyr-17 in HP-51. All four tyrosines are essential for maintaining the structure of the isolated headpiece, and Tyr-7, -12, and -17 appear to be stacked.

Escherichia coli↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 2. Partial analysis of the aliphatic region of the Lac repressor headpiece spectrum.

The 360-MHz 1H NMR spectrum of native lac repressor headpiece (HP-51 or HP-59) contains a large number (greater than 30%) of aliphatic side-chain methyl and backbone alpha-CH resonances and three of four aromatic tyrosine multiplet resonances shifted to high-field chemical shift positions, indicating the presence of extensive folded structure. Denaturation leads to loss of the NMR chemical shift differences. Resonance identifications of the 27 methyl-possessing amino acids in HP-59 have been made by using resolution enhancement, double-resonance, and difference spectra. There are three firmly assigned methyl resonances and 21 pairwise identifications of methyl resonances in HP-51. Comparison of HP-51 and HP-59 allows identification of four additional methyl groups in amino acid residues 52--59. The sequence HP-50--59 is not essential to maintain the structure of HP-59, but it is of interest itself as the flexible hinge portion connecting HP to the tetrameric core of whole repressor.

Escherichia coli↗