1H nuclear magnetic resonance assignments and secondary structure of porcine C5ades Arg

Sequence-specific assignments of the 1H nuclear magnetic resonance spectrum of porcine C5ades Arg are described. Assignments were facilitated by comparison of spectra obtained in H2O with partially exchanged spectra obtained in 2H2O. The sequence-specific assignments thus obtained were used to characterized the regular secondary structure in the protein, which is helical in the regions 2 to 11, 16 to 27, 35 to 41 and 45 to 64. The structure is very similar to that of human and bovine C5a.     

1H nuclear magnetic resonance assignments for d-(GCATTAATGC)2 using experimental refinements of established procedures

Sequence-specific 1H nuclear magnetic resonance assignments are presented for d-(GCATTAATGC)2. Using omega 1-scaled double quantum-filtered correlated spectroscopy, two-quantum spectroscopy, relayed coherence transfer spectroscopy and detailed analysis of the fine structure in these phase-sensitive spectra, the spin system of the bases and deoxyribose rings were identified entirely via scalar proton-proton couplings. The sequential connectivities were established with two-dimensional nuclear Overhauser enhancement spectra recorded with a short mixing time of 60 milliseconds. These spectra contain only a small number of cross-peaks, corresponding to the shortest proton-proton distances prevailing in the DNA. They are thus easy to interpret, and therefore the presently proposed modifications of the established assignment procedures should enable studies of larger DNA duplexes with intrinsically more complex nuclear magnetic resonance spectra, and they also provided an improved basis for conformational studies of DNA fragments.     

1H, 13C and 15N resonance assignments for Binder of Arl2, BART

We report ¹H, ¹³C and ¹⁵N resonance assignments for Binder of Arl Two (BART), an effector of the small G protein Arl2. The BMRB accession code is 15914.     

Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by (13)C and (31)P nuclear magnetic resonance

The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle.     

1H, 13C, and 15N resonance assignments of human phosphohistidine phosphatase 1 (PHPT1)

Phosphohistidine phosphatase 1 (PHPT1) is the first protein histidine phosphatase identified in vertebrates. The NMR assignments of human PHPT1 are essential for solution structure determination and NMR study of the protein interactions of PHPT1 with its potential substrates.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.     

Backbone 1H, 13C and 15N resonance assignments of human vaccinia-related kinase 1 (VRK1)

Vaccinia-related kinase 1 (VRK1) is one of the mitotic kinases which play key roles in cell cycle control and chromatin modifications. To understand the biological role of the kinase and gain insights into its catalytic mechanism, we performed NMR assignments of catalytically active form of VRK1 with 361 amino acids residues. Here, we present the backbone NMR resonance assignments of the kinase.     

Sequential1H and13C resonance assignments for an octa- and decasaccharide of theN-acetyllactosamine type by multiple-step relayed correlation and heteronuclear correlation nuclear magnetic resonance

400 MHz¹H-NMR and 100 MHz¹³C-NMR spectra of a neutral octasaccharide and of a disialyldecasaccharide of theN-acetyllactosamine type were studied. The resonance assignments were made by combining multiple-relayed coherence-transfer chemical-shift-correlated spectroscopy (multiple-RELAY-COSY) and¹H/¹³C-shift correlated 2D experiments. The complete analysis of the¹H and¹³C spectra was performed.     

Complete sequence-specific 1H nuclear magnetic resonance assignments for mouse epidermal growth factor

The 1H NMR spectrum of the mouse epidermal growth factor (53 residues) was analyzed with the use of two-dimensional NMR techniques. All the observable 296 proton resonances were completely assigned in a sequential manner. For the spin system identification, two-dimensional homonuclear Hartmann-Hahn spectrum was useful, especially for arginine and proline residues. The easy spin system identification of these long-side-chain-bearing amino acid residues greatly facilitated the sequence-specific resonance assignment of the epidermal growth factor.     

Backbone and side-chain 1H, 13C, 15N resonance assignments of rat lipocalin2

Lipocalin2 plays an important role in the innate immune system. In this article we report the backbone and side-chain resonance assignments of rat lipocalin2 (rLcn2). These assignments provide a basis for determining the structure and dynamics of rLcn2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: 31P nuclear magnetic resonance study

31P nuclear magnetic resonance (NMR) has been used to study the 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP). Comparison of the proton-decoupled spectra of 5-phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) and the SP diastereomer of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) with the parent molecule revealed a characteristic large downfield chemical shift change for the resonance signal associated with the thiophosphate group (delta delta approximately 40-50 ppm) and an increase in the magnitude of the phosphate-thiophosphate spin-spin coupling constant (delta J alpha beta approximately 10 Hz). Both these changes are consistent with the observed effects of sulfur substitution on the behavior of the adenosine nucleotides, particularly ADP [Jaffe, E. K., & Cohn, M. (1978) Biochemistry 17, 652-657]. High-field 31P NMR has also been used to demonstrate the diastereomeric purity of P-Rib-PP alpha S (Sp diastereomer) and the greater lability of this analogue when compared with both P-Rib-PP beta S and P-Rib-PP. Sulfur substitution was found to cause a large decrease in the apparent pKa associated with the thiophosphate moiety of P-Rib-PP beta S (delta pKa approximately 1.4 units) and also to enhance the sensitivity of the thiophosphate chemical shift to protonation and, in particular, to Mg2+ binding, compared with P-Rib-PP. The potential application of the phosphorothioate analogues as probes of the reactions catalyzed by the phosphoribosyltransferase enzymes is discussed.     

In vivo 31P and 13C nuclear magnetic resonance studies of acetate metabolism in Chromatium vinosum

31P and 13C nuclear magnetic resonance (NMR) experiments were performed on suspensions of the phototrophic bacterium Chromatium vinosum incubated anaerobically in the dark. 31P NMR spectra revealed that during prolonged dark incubation high ATP levels are maintained. This phenomenon was independent of the presence of the energy reserves polyglucose and polyphosphate. 13C NMR experiments revealed that the amino acids glutamate, aspartate, and alanine are the major products of acetate incorporation in the dark. Apart from these amino acids, poly-beta-hydroxybutyrate was also formed. Acetate metabolism was markedly stimulated by the presence of polyglucose. The specific 13C activity of glutamate C-2 was approximately 50% that of glutamate C-4. The idea is discussed that this difference is the consequence of the maintenance of redox balance during entry of acetate into cell metabolism.