Characterization of pheophytin ground states in Rhodobacter sphaeroides R26 photosynthetic reaction centers from multispin pheophytin enrichment and 2-D 13C MAS NMR dipolar correlation spectroscopy

The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from C-13 chemical shift data, A new experimental approach involving multispin C-13 labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly C-13 biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state C-13 magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state C-13-C-13 correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many C-13 resonances of the [U-C-13]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of C-13 shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.Solid state NMR/Biophysical Organic ChemistryBiological and Soft Matter Physic

Monitoring of biomass composition from microbiological sources by means of FT-IR spectroscopy.

An FT-IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm(-1). Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the C-H stretching vibrational population between 2,984 and 2,780 cm(-1). Carbohydrate content was determined using integration over a C-O and C-O-C stretching band area between 1,180 and 1,133 cm(-1). Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra-batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT-IR spectroscopy compared well with the results obtained from elemental analysis

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium

Item does not contain fulltextUniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for (15)N and (13)C,(15)N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells

Characterisation of uniformly C-13, N-15 labelled bacteriochlorophyll a and bacteriopheophytin a in solution and in solid state: complete assignment of the C-13, H-1 and N-15 chemical shifts

Solid state NMR/Biophysical Organic Chemistr

Carbon conversion and metabolic rate in two marine sponges

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.

A set-up to study photochemically induced dynamic nuclear polarization in phtosynthetic reaction centres by solid-state NMR

418-423Recently, solid-state NMR spectroscopy became a viable method to investigate photosynthetic reaction centres (RCs) on t e atomic level. To study the electronic structure of the radical cation state of the RC, occurring after the electron emission, solid-state NMR using an illumination set-up can be exploited. This paper describes the illumination set-up we designed for a standard Bruker wide-bore MAS NMR probe. In addition we demonstrate its application to get information from the  active site in photosynthetic reaction centres of Rhodobacter spaeroides R-26 by photochemically induced dynamic nuclear polarization (photo-CIDNP). Solid-state NMR spectra of natural abundance 13C in detergent solubilized quinone depleted photosynthetic reaction centres under continuous illumination showed exceptionally strong nuclear spin polarization in NMR lines. Both enhanced-absorptive and emissive polarization were seen in the carbon spectrum which could be assigned to a bacteriochlorophyll a (BChl a) cofactor, presumably the special pair BChl a. The sign and intensities of the 13C NMR signals provide information about the electron spin density distribution of the transiently formed radical P.+ on the atomic level

Efficient conditions for the photoaccumulation of H-A(-) in the photosynthetic reaction centre of Rhodobacter sphaeroides R26 with uniformly labelled bacteriopheophytin monitored by Fourier transform infrared difference spectroscopy

Biological and Soft Matter Physic