Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
(49/1209)
Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa. (+info)
Flavin-protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C- and 15N-labelled flavin.
(50/1209)
A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the pi-electron distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins. With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane. In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme. (+info)
Using stable isotope natural abundances (delta 15N and delta 13C) to integrate the stress responses of wild barley (Hordeum spontaneum C. Koch.) genotypes.
(51/1209)
To integrate the complex physiological responses of plants to stress, natural abundances (delta) of the stable isotope pairs 15N/14N and 13C/12C were measured in 30 genotypes of wild barley (Hordeum spontaneum C. Koch.). These accessions, originating from ecologically diverse sites, were grown in a controlled environment and subjected to mild, short-term drought or N-starvation. Increases in total dry weight were paralleled by less negative delta 13C in shoots and, in unstressed and droughted plants, by less negative whole-plant delta 13C. Root delta 15N was correlated negatively with total dry weight, whereas shoot and whole-plant delta 15N were not correlated with dry weight. The difference in delta 15N between shoot and root varied with stress in all genotypes. Shoot-root delta 15N may be a more sensitive indicator of stress response than shoot, root or whole-plant delta 15N alone. Among the potentially most productive genotypes, the most stress-tolerant had the most negative whole-plant delta 15N, whether the stress was drought or N-starvation. In common, controlled experiments, genotypic differences in whole-plant delta 15N may reflect the extent to which N can be retained within plants when stressed. (+info)
Weather and nodule mediated variations in delta 13C and delta 15N values in field-grown soybean (Glycine max L.) with special interest in the analyses of xylem fluids.
(52/1209)
The nodulating soybean (Enrei) and its non-nodulating mutant (EN 1282) were grown in outdoor plots for 2 years (1994: extraordinary dry, high temperature, 1995: ordinary year). Carbon and nitrogen accumulation, delta 13C and delta 15N values in plant parts and xylem fluids and delta 15N values in the water-extractable soil N were analysed throughout the growing period. Plant growth in 1994 was rapid during the early growth stages, but no pods were produced. In 1995, plant growth was normal and pods were formed. The delta 13C values of the leaves were less negative in 1994 than in 1995 and the nodulated plants showed less negative delta 13C values than non-nodulated plants in both years. The delta 13C values of the leaves during the vegetative phase were positively correlated to the leaf N concentrations. Leaf N concentrations in their turn were influenced by nodulation and weather conditions and/or soil available N. The delta 15N values in the plants and xylem fluids were lower in the nodulated soybean than in non-nodulated soybean in both years, and estimates of the contribution of N2 fixation in nodulated plants based on plant top delta 15N values were 7-14% in 1994 and 37-63% in 1995. The delta 13C values of xylem fluids did not differ between nodulated and non-nodulated plants. Thus, the expected contribution by phosphopenolpyruvate carboxylase-mediated CO2 fixation in the root nodules to plant C-incorporation could not have been significant. (+info)
Effect of hypoglycemia on amino acid and protein metabolism in healthy humans.
(53/1209)
In response to hypoglycemia, healthy individuals rapidly antagonize insulin action on glucose and lipid metabolism, but the effects on protein metabolism are unclear. Because amino acids are an important substrate for gluconeogenesis and a fuel alternative to glucose for oxidation, we evaluated whether hypoglycemia antagonizes the hypoaminoacidemic and the antiproteolytic effects of insulin and changes the de novo synthesis of glutamine, a gluconeogenic amino acid. To this purpose, in 7 healthy subjects, we performed 2 studies, 3.5 h each, at similar insulin but different glucose concentrations (i.e., 4.9 +/- 0.1 mmol/l [euglycemic clamp] or 2.9 +/- 0.2 mmol/l [hypoglycemic clamp]). As expected, hypoglycemia antagonized the insulin suppression of glucose production achieved in euglycemia (from 21 +/- 15 to 116 +/- 12% of basal, P < 0.001), the stimulation of glucose uptake (from 207 +/- 28 to 103 +/- 7% of basal, P < 0.01) and the suppression of circulating free fatty acids (from 30 +/- 5 to 80 +/- 17% of basal, P < 0.001). In contrast, hypoglycemia increased the insulin suppression of circulating leucine (from 63 +/- 1 to 46 +/- 2% of basal, P < 0.001) and phenylalanine (from 79 +/- 3 to 64 +/- 3% of basal, P < 0.001) concentrations. Hypoglycemia did not change the insulin suppression of proteolysis (from 79 +/- 2 to 82 +/- 4% of basal, P < 0.001). However, hypoglycemia doubled the insulin suppression of the glutamine concentrations (from 84 +/- 3 to 63 +/- 3% of basal, P < 0.01) in the absence of significant changes in the glutamine rate of appearance, but it also caused an imbalance between glutamine uptake and release. This study demonstrates that successful counterregulation does not affect proteolysis. Moreover, it does not increase the availability of circulating amino acids by de novo synthesis. In contrast, despite the lower concentration of circulating amino acids, hypoglycemia increases the uptake of glutamine that can be used for gluconeogenesis and as a fuel alternative to glucose. (+info)
A reverse flow-metabolism mismatch pattern: a new marker of viable myocardium with greater contractility during dobutamine stress than myocardium with a flow-metabolism mismatch pattern.
(54/1209)
Few studies have investigated the contractility of myocardium with a reverse flow-metabolism pattern; that is, greater uptake of nitrogen- 13-ammonia (NH3) than fluorine- 18-fluorodeoxyglucose (FDG) on positron emission tomography (PET). This study examined the contraction thickening represented by count increase in ECG-gated FDG-PET of myocardium with a reverse flow-metabolism pattern during low-dose dobutamine stress. Fifty-four patients with myocardial infarction were studied. Relative NH3 and FDG uptake (%NH3, %FDG) and %count increase were measured in 216 apical and 216 lateral segments on ECG-gated FDG-PET. The %count increase during low-dose dobutamine stress was greater in myocardium with a reverse flow-metabolism mismatch pattern than in myocardium with a flow-metabolism mismatch pattern (35.9+/-25.7% vs 24.6+/-15.9%, p=0.0221 in apical segments, and 38.4+/-22.6% vs 27.6+/-18.4%, p=0.0040 in lateral segments) despite smaller %FDG. A reverse flow-metabolism mismatch pattern should be noted as a new marker of viable myocardium with greater contractility during dobutamine stress than myocardium with a flow-metabolism mismatch pattern. (+info)
Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging.
(55/1209)
The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered. (+info)
Compartmental modeling with nitrogen-15 to determine effects of degree of fat saturation on intraruminal N recycling.
(56/1209)
Two- and three-compartment models were developed to describe N kinetics within the rumen using three Holstein heifers and one nonlactating Holstein cow fitted with ruminal and duodenal cannulas. A 4 x 4 Latin square design included a control diet containing no supplemental fat and diets containing 4.85% of diet dry matter as partially hydrogenated tallow (iodine value = 13), tallow (iodine value = 51), or animal-vegetable fat (iodine value = 110). Effects of fat on intraruminal N recycling and relationships between intraruminal N recycling and ruminal protozoa concentration or the efficiency of microbial protein synthesis were determined. A pulse dose of 15(NH4)2SO4 was introduced into the ruminal NH3 N pool, and samples were taken over time from the ruminal NH3 N and nonammonia N pools. For the three-compartment model, precipitates of nonammonia N after trichloroacetic acid and ethanol extraction were defined as slowly turning over nonammonia N; rapidly turning over nonammonia N was determined by difference. Curves of 15N enrichment were fit to models with two (NH3 N and nonammonia N) or three (NH3 N, rapidly turning over nonammonia N, and slowly turning over nonammonia N) compartments using the software SAAM II. Because the three-compartment model did not remove a small systematic bias or improve the fit of the data, the two-compartment model was used to provide measurements of intraruminal N recycling. Intraruminal NH3 N recycling (45% for control) decreased linearly as fat unsaturation increased (50.2, 43.0, and 41.7% for partially hydrogenated tallow, tallow, and animal-vegetable fat, respectively). Intraruminal nitrogen recycling was not correlated with efficiency of microbial protein synthesis or ruminal protozoa counts. (+info)