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Query: UNIPROT:Q9UMR3 (NMR)
 150,598 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Cepsilon methyl group of the 2 methionine residues in sperm whale myoglobin was enriched with respect to 13C. This was accomplished by treatment of the apomyoglobin at pH 4 at room temperature with a 100-fold proportion of 13CH3I to form an intermediate containing enriched S-methylmethionine. Unselective demethylation to regain the apomyoglobin structure was accomplished by treatment at pH 10.5 with 0.5 M dithioerythritol at 37 degrees for 18 h. Reagents were removed at each stage by dialysis against dilute sodium azide solution. Hemin was reincorporated to form the holoprotein in a way that avoided the presence of an excess of the small molecule. After chromatographic purification the enriched myoglobin was obtained in a yield of between 29 and 60%. The composition, absorbance spectrum, circular dichroism spectrum, isoionic point, electrophoretic behavior, and oxygen-binding behavior following reduction were all indistinguishable from those of the virgin protein. NMR measurements were made at 15.1, 25.2, and 67.9 MHz at 27-30 degrees. The two enriched loci are represented by separate resonances that appear slightly downfield of the spectral position of the corresponding resonance in free methionine. The positions of these resonances are sensitive to pH and to the ligand bound at the heme group which is approximately 17 A distant from each methionine Cepsilon. On the basis of two separate types of experiment the downfield resonance was assigned to methionine 55 and the upfield resonance to methionine 131. Part of the observed variations in chemical shift could be treated as arising from pseudocontact interactions but part was ascribed to structural changes communicated to the environment of each methionine residue as a result of changes in heme ligand, pH, or temperature. The linewidths of the methionine Cepsilon resonances are narrowed by increasing temperature according to an Arrhenius energy of activation of nearly 3 kcal. The spin-lattice relaxation times, T1, of the two methionine Cepsilon resonances at the three spectrometer frequencies were interpreted to indicate the existence of rotational motions in each side chain in addition to that about the Sdelta-Cepsilon bond. The results as a whole show that the two methionine side chains undergo continuous variations in environment, and that these variations are controlled by events at a distance within the protein structure. It is suggested that the structural lability serves the function of facilitating conformational variations and adjustments within the heme pocket.
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PMID:Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups. 1 65

The 31P high resolution NMR spectra of concentrated suspensions of Escherichia coli cells have been measured at 145.8 MHz. The position of the orthophosphate resonance is used as a measure of internal and external pH. In accord with Paddan, Zilberstein and Rottenberg ((1976) Eur. J. Biochem. 63, 533--541) it is shown that when properly energized the internal pH is 7.5 +/- 0.1. By synchronizing the NMR data acquisition with 3-s bursts of O2 it is possible to measure the internal pH with a time resolution of about 1 s. It is shown that at 20 degrees C the pH remains constant for times longer than 15 s after the oxygen is discontinued and it decays in several minutes.
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PMID:On the measurement of pH in Escherichia coli by 31P nuclear magnetic resonance. 2 81

The 270 MHz 1H-NMR spectrum of soya bean lipoxygenase-1 (linoleate: oxygen oxidoreductase, EC 1.13.11.12) was investigated at 298 K and at several pH values. A large fraction of the protein (50%) was found to be effectively random coil.
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PMID:Proton NMR studies on soya bean lipoxygenase-1. 2 52

A model is presented of the pH-dependence of the number of oxygen-linked chloride binding sites established by nuclear magnetic resonance quadrupole-relaxation studies on various mutant and chemically modified hemoglobins. The predictions of the model are in good qualitative agreement with the measured pH-dependences of the linewidth of the 35Cl- NMR signal. The obtained agreement implies that more chloride is bound to oxygenated than to deoxygenated hemoglobin.
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PMID:A model of the pH-dependence of the number of oxygen-linked chloride binding sites in hemoglobin. 3 34

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.
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PMID:Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies. 3 20

Adenosine 5'-(thiophosphate) AMPS) contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions. A general method was developed to distinguish between the "pro-R" and "pro-S" oxygens. When we converted the AMPS to the isomer A of adenosine 5'-(1-thiotriphosphate) (ATPalphaS), which is known to have S configuration at Palpha, the pro-R oxygen is incorporated into the bridge position, whereas the pro-S oxygen is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge oxygens based on the decrease in the peak intensity of 31P NMR signals caused by the directly bound 17O isotope. The method was used to elucidate the stereochemical course of acetate activation catalyzed by yeast acetyl coenzyme A (CoA) synthetase. The results indicate that yeast acetyl-CoA synthetase is specific for the isomer B of ATPalphaS and that the nucleophilic displacement proceeds with net inversion of configuration at Palpha of ATPalphaS (B), supporting the "in-line" mechanism.
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PMID:Use of phosphorus-31 nuclear magnetic resonance to distinguish bridge and nonbridge oxygens of oxygen-17-enriched nucleoside triphosphates. Stereochemistry of acetate activation by acetyl coenzyme A synthetase. 3 27

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.
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PMID:Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae. 4 May 90

An equilibrium mixture of highly enriched [18(O)]Pi (represents the mixture of [[18(O)4]Pi, [[18(O)3]Pi, [18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-[16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-[18(O)4]-phosphate and of [16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the [18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom.
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PMID:Purine nucleoside phosphorylase cleaves the C--O bond of ribose 1-phosphate. Evidence from the 18O shift in 31P NMR. 10 57

The structure of the leuco compound of the purple dye which is formed in mixtrues of N,N-dimethylaniline-N-oxide (DANO) and ferrihemoglobin or ferricytochrome c was elucidated. IR, NMR, mass spectroscopy, and synthesis by oxidation of mixtures of N-methylaniline and 2-dimethylaminophenol showed that the leuco compound is produced by condensation of these two compounds. But only X-ray analysis proved the structure: 2-dimethylamino-4-(N-methylanilino)-phenol. The purple dye was produced from the leuco compound by withdrawal of two electrons and may be considered as resonance hybrid of the p-quinonimine and the o-quinonimine. When DANO was incubated with ferrihemoglobin or ferricytochrome c the oxygen of DANO was used for the production of the dye by oxidation of N-methylaniline and 2-dimethylaminophenol. The amount of N,N-dimethylaniline found in the incubation mixtures corresponded with the amount of purple dye produced. In the absence of molecular oxygen from incubation mixtures of DANO with cytochrome c the purple dye was formed at the same rate as under air. In blood in vitro the purple dye catalytically transferred electrons from ferrohemoglobin to molecuar oxygen. Its ferrihemoglobin-forming activity was lower than that of 4-dimethylaminophenol but higher than that of 2-dimethylaminophenol. The chemical mechanism of the autocatalytic formation of ferrihemoglobin by DANO is described.
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PMID:Mechanism of the autocatalytic formation of ferrihemoglobin by N,N-dimethylaniline-N-oxide. Structure and ferrihemoglobin forming activity of the purple dye. 17 65

The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.
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PMID:A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label. 18 97


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