176 research outputs found
Residual backbone and side-chain 13C and 15N resonance assignments of the intrinsic transmembrane light-harvesting 2 protein complex by solid-state Magic Angle Spinning NMR spectroscopy
This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion C-13-C-13 correlation experiments with mixing times of 20 and 500 ms and band selective C-13-C-13 correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long alpha-helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the C-13-C-13 correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins.Solid state NMR/Biophysical Organic Chemistr
Cyanosis, hemolysis, decreased HbA1c and abnormal co-oximetry in a patient with hemoglobin M Saskatoon [HBB:c.190C > T p.His64Tyr]
We describe a first Dutch case of Hb M Saskatoon (HBB:c.190C > T p.His64Tyr) in a 47-year-old female Dutch patient who presented with cyanosis, hemolysis, and abnormal co-oximetry. A mean corpuscular volume (MCV) of 105 fL caused by reticulocytosis (160 x 10(9)/L) and low red blood cell count (3.6 x 10(12)/L) suggested an increased erythrocyte turnover. An HPLC glyco-globin analysis revealed a decreased HbA1c fraction of 12.3 mmol/mmol, HbA0 of 93.3% and an additional unidentified fraction at 1.2 min. DNA sequencing revealed a missense mutation in the HBB gene, (HBB:c.190C > T p.His64Tyr), known as Hb M Saskatoon, a variant which has been previously identified as an unstable hemoglobin variant leading to methemoglobinemia and anemia. In this report, we describe the clinical and remarkable laboratory aspects of our patient with Hb M Saskatoon, and the consequences for treatment and drug use.Genetics of disease, diagnosis and treatmen
Selective chemical shift assignment of bacteriochlorophyll a in uniformly [C-13-N-15]-labeled light-harvesting 1 complexes by solid-state NMR in ultrahigh magnetic field
Solid state NMR/Biophysical Organic Chemistr
Solid-state NMR applied to photosynthetic light-harvesting complexes
This short review describes how solid-state NMR has provided a mechanistic and electronic picture of pigment–protein and pigment–pigment interactions in photosynthetic antenna complexes. NMR results on purple bacterial antenna complexes show how the packing of the protein and the pigments inside the light-harvesting oligomers induces mutual conformational stress. The protein scaffold produces deformation and electrostatic polarization of the BChl macrocycles and leads to a partial electronic charge transfer between the BChls and their coordinating histidines, which can tune the light-harvesting function. In chlorosome antennae assemblies, the NMR template structure reveals how the chromophores can direct their self-assembly into higher macrostructures which, in turn, tune the light-harvesting properties of the individual molecules by controlling their disorder, structural deformation, and electronic polarization without the need for a protein scaffold. These results pave the way for addressing the next challenge, which is to resolve the functional conformational dynamics of the lhc antennae of oxygenic species that allows them to switch between light-emitting and light-energy dissipating states
Nuclear magnetic resonance secondary shifts of a light-harvesting 2 complex reveal local backbone perturbations induced by its higher-order interactions
Solid state NMR/Biophysical Organic Chemistr
Association of Altered Plasma Lipidome with Disease Severity in COVID-19 Patients
The severity of COVID-19 is linked to an imbalanced immune response. The dysregulated metabolism of small molecules and bioactive lipids has also been associated with disease severity. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyze over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). This is the third publication in a series, and it reports the results of comprehensive lipidome profiling using targeted LC-MS/MS. We identified 1076 lipid features across 25 subclasses, including glycerophospholipids, sterols, glycerolipids, and sphingolipids, among which 531 lipid features were dramatically changed in the plasma of intensive care unit (ICU) patients compared to patients in the ward. Patients in the ICU showed 1.3–57-fold increases in ceramides, (lyso-)glycerophospholipids, diglycerides, triglycerides, and plasmagen phosphoethanolamines, and 1.3–2-fold lower levels of a cyclic lysophosphatidic acid, sphingosine-1-phosphates, sphingomyelins, arachidonic acid-containing phospholipids, lactosylceramide, and cholesterol esters compared to patients in the ward. Specifically, phosphatidylinositols (PIs) showed strong fatty acid saturation-dependent behavior, with saturated fatty acid (SFA)- and monosaturated fatty acid (MUFA)-derived PI decreasing and polystaturated (PUFA)-derived PI increasing. We also found ~4000 significant Spearman correlations between lipids and multiple clinical markers of immune response with |R| ≥ 0.35 and FDR corrected Q < 0.05. Except for lysophosphatidic acid, lysophospholipids were positively associated with the CD4 fraction of T cells, and the cytokines IL-8 and IL-18. In contrast, sphingosine-1-phosphates were negatively correlated with innate immune markers such as CRP and IL-6. Further indications of metabolic changes in moderate COVID-19 disease were demonstrated in recovering ward patients compared to those at the start of hospitalization, where 99 lipid species were altered (6 increased by 30–62%; 93 decreased by 1.3–2.8-fold). Overall, these findings support and expand on early reports that dysregulated lipid metabolism is involved in COVID-19.</p
Safety of D-ribose as a novel food pursuant to Regulation (EU) 2015/2283
Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on D-ribose as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The applicant intends to market the NF as ingredient in a variety of foods, food supplements and in certain foods for specific groups. The NF is produced by fermentation using a transketolase-deficient strain of Bacillus subtilis and marketed as Bioenergy Ribose (TM). The information provided on the batch-to-batch variability, specifications, stability, production process and history of the organism used as a source of the NF is sufficient and does not raise safety concerns. The Panel considers that the effects observed in a subchronic toxicity study in rats could be the consequence of nutritional imbalances, but toxicological effects could not be ruled out; from this study, the Panel derived a No observed adverse effect level (NOAEL) of 3.6 g/kg body weight (bw) per day. From the human studies indicating a potential decrease in glucose levels and/or the occurrence of transient symptomatic hypoglycaemia at intakes of 10 g of D-ribose, the Panel defined 70 mg/kg bw per day as the NOAEL with respect to hypoglycaemia that can be considered applicable for adults. For children, the Panel acknowledges the lack of human data directly relevant for this population group. Based on the NOAEL derived from the subchronic toxicity study in rats, an acceptable level of intake of 36 mg/kg bw per day was defined that would also take into account the potentially increased sensitivity of certain population groups to hypoglycaemia. The Panel concludes that the NF is safe for the general population at intake levels up to 36 mg/kg bw per day and considers that the safety of the NF at the intended uses and use levels as proposed by the applicant has not been established. (C) 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.Peer reviewe
Multiple segmental and selective isotope labeling of large RNA for NMR structural studies
Multiple segmental and selective isotope labeling of RNA with three segments has been demonstrated by introducing an RNA segment, selectively labeled with 13C9/15N2/2H(1′, 3′, 4′, 5′, 5′′)-labeled uridine residues, into the central position of the 20 kDa ε-RNA of Duck Hepatitis B Virus. The RNA molecules were produced via two efficient protocols: a two-step protocol, which uses T4 DNA ligase and T4 RNA ligase 1, and a one-pot protocol, which uses T4 RNA ligase 1 alone. With T4 RNA ligase 1 all not-to-be-ligated termini are usually protected to prevent formation of side products. We show that such labor-intensive protection of termini is not required, provided segmentation sites can be chosen such that the segments fold into the target structure or target-like structures and thus are not trapped into stable alternate structures. These sites can be reliably predicted via DINAMelt. The simplified NMR spectrum provided evidence for the presence of a U28 H3-imino resonance, previously obscured in the fully labeled sample, and thus of the non-canonical base pair U28:C37. The demonstrated multiple segmental labeling protocols are generally applicable to large RNA molecules and can be extended to more than three segments
- …