One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C, 15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the alpha-helical segments of the protein, mostly found in the membrane interior.
In this investigation we report a complete assignment of (13)C, (1)H and (15)N solution and solid state chemical shifts of two bacterial photosynthetic pigments, bacteriochlorophyll (BChl) a and bacteriopheophytin (BPheo) a. Uniform stable-isotope labelling strategies were developed and applied to biosynthetic preparation of photosynthetic pigments, namely uniformly (13)C, (15)N labelled BChl a and BPheo a. Uniform stable-isotope labelling with (13)C, (15)N allowed performing the assignment of the (13)C, (15)N and (1)H resonances. The photosynthetic pigments were isolated from the biomass of photosynthetic bacteria Rhodopseudomonas palustris 17001 grown in uniformly (13)C (99%) and (15)N (98%) enriched medium. Both pigments were characterised by NMR in solution (acetone-d(6)) and by MAS NMR in solid state and their NMR resonances were recorded and assigned through standard liquid 2D (13)C-(13)C COSY, (1)H-(13)C HMQC, (1)H-(15)N HMBC and solid 2D (13)C-(13)C RFDR, (1)H-(13)C FSLG HETCOR and (1)H-(15)N HETCOR correlation techniques at 600 MHz and 750 MHz. The characterisation of pigments is of interest from biochemical to pharmaceutical industries, photosynthesis and food research.
The electronic ground states of the bacteriochlorophyll a type B800 and type B850 in the light-harvesting 2 complex of Rhodopseudomonas acidophila strain 10050 have been characterized by magic angle spinning (MAS) dipolar (13)C-(13)C correlation NMR spectroscopy. Uniformly [(13)C,(15)N] enriched light-harvesting 2 (LH2) complexes were prepared biosynthetically, while [(13)C,(15)N]-B800 LH2 complexes were obtained after reconstitution of apoprotein with uniformly [(13)C,(15)N]-enriched bacteriochlorophyll cofactors. Extensive sets of isotropic (13)C NMR chemical shifts were obtained for each bacteriochlorin ring species in the LH2 protein. (13)C isotropic shifts in the protein have been compared to the corresponding shifts of monomeric BChl a dissolved in acetone-d(6). Density functional theory calculations were performed to estimate ring current effects induced by adjacent cofactors. By correction for the ring current shifts, the (13)C shift effects due to the interactions with the protein matrix were resolved. The chemical shift changes provide a clear evidence for a global electronic effect on the B800 and B850 macrocycles, which is attributed to the dielectrics of the protein environment, in contrast with local effects due to interaction with specific amino acid residues. Considerable shifts of -6.2 < Deltasigma < +5.8 ppm are detected for (13)C nuclei in both the B800 and the B850 bacteriochlorin rings. Because the shift effects for the B800 and B850 are similar, the polarization of the electronic ground states induced by the protein environment is comparable for both cofactors and corresponds with a red shift of approximately 30 nm relative to the monomeric BChl dissolved in acetone-d(6). The electronic coupling between the B850 cofactors due to macrocycle overlap is the predominant mechanism behind the additional red shift in the B850.
The carbon metabolism of two marine sponges, Haliclona oculata and Dysidea avara, has been studied using a 13C isotope pulse-chase approach. The sponges were fed 13C-labeled diatoms (Skeletonema costatum) for 8 h and they took up between 75 and 85%. At different times, sponges were sampled for total 13C enrichment, and fatty acid (FA) composition and 13C enrichment. Algal biomarkers present in the sponges were highly labeled after feeding but their labeling levels decreased until none was left 10 days after enrichment. The sponge-specific FAs incorporated 13C label already during the first day and the amount of 13C label inside these FAs kept increasing until 3 weeks after labeling. The algal-derived carbon captured by the sponges during the 8-h feeding period was thus partly respired and partly metabolized during the weeks following. Apparently, sponges are able to capture enough food during short periods to sustain longer-term metabolism. The change of carbon metabolic rate of fatty acid synthesis due to mechanical damage of sponge tissue was studied by feeding sponges with 13C isotope–labeled diatom (Pheaodactylum tricornutum) either after or before damaging and tracing back the 13C content in the damaged and healthy tissue. The filtration and respiration in both sponges responded quickly to damage. The rate of respiration in H. oculata reduced immediately after damage, but returned to its initial level after 6 h. The 13C data revealed that H. oculata has a higher metabolic rate in the tips where growth occurs compared to the rest of the tissue and that the metabolic rate is increased after damage of the tissue. For D. avara, no differences were found between damaged and non-damaged tissue. However, the filtration rate decreased directly after damage.
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