Determination of the pigment stoichiometry of the photochemical reaction center of photosystem II

AbstractThe stoichiometry of chlorophyll a, pheophytin α and β-carotene in the photochemical reaction center of Photosystem II was analyzed by reversed phase high-performance liquid chromatography (HPLC) with methanol as the mobile phase, and by the shape of spectra of extracts in 80% acetone. For the HPLC method the molar extinction coefficient of pheophytin a in methanol was redetermined, while for the spectroscopic method spectra of extracts in 80% acetone were simulated by fitting with spectra of isolated chlorophyll a, pheophytin a and β-carotene in 80% acetone. Both methods give internally consistent results, and suggest that the reaction center of Photosystem II isolated by a short Triton X-100 treatment binds 6 chlorophyll a per 2 pheophytin a molecules. We also present evidence that prolonged exposure of the Photosystem II reaction center complex to Triton X-100 does not result in the loss of chlorophyll from the complex. Based on a comparison with spectra reported in publications from other groups, we conclude that the chlorophyll to pheophytin ratio has previously been underestimated to sometimes very significant extents, and that, as yet, no Photosystem II reaction center particles have been purified that bind less than 5–6 chlorophyll a per 2 pheophytin a

Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes

We present an extended analysis of the organization of green plant photosystem II and its associated light-harvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII-LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry 38, 2233-2239], the following results were obtained. The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about -20 degrees and -50 degrees, respectively. The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions. A new type of megacomplex was observed with a characteristic windmill-like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips. Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins

Characterization by electron microscopy of dimeric Photosystem II core complexes from spinach with and without CP43

Dimeric associations of the D1-D2-CP47 and D1-D2-CP47-CP43 complexes of Photosystem II from spinach were isolated and purified with sucrose density gradient centrifugation and gel filtration chromatography and analyzed by electron microscopy and image analysis. Images of both preparations show characteristic details in protein density. The location of the CP43 subunit and the way the dimers are associated could be determined from a comparison between diamond-like monomeric projections of the D1-D2-CP47-CP43 complex (maximal dimensions along the diagonals 10–12 and 7–8 nm) and triangle-like monomeric projections of the D1-D2-CP47 complex (dimensions 8–9 and 7–8 nm). Both isolated complexes have different dimeric configurations than observed before in several other dimeric complexes, and based on biochemical considerations we conclude that both newly observed configurations are artificial. The observation of the artificial aggregates, however, allows conclusions on the organization of Photosystem II in two-dimensional crystals and on the size of the monomeric unit. We propose a model for the location of D1, D2, CP43 and CP47 in the Photosystem II core complex in which CP43 and CP47 are positioned at the tips of the monomeric unit, closely connected to D2 and D1, respectively.

Numerical Optimization of Underactuated Flexure-Based Grippers

Robotic manipulation in the agri-food industry faces several issues, including object variation, fragility and food safety. Underactuated flexure-based gripper allow passive adaptation to object variation, whilst monolithic flexure joints drive down cost, part-count, hygiene requirements, contamination and wear. However, designing flexure-based grippers presents challenges in achieving sufficient support stiffness, load-bearing capacity and joint deflection. Additionally, modeling the non-linear flexure behavior may become computationally expensive, especially under wide a variety of load cases, limiting the optimization approaches to simple structures and joints. In this work we present an interleaved computational optimization algorithm for underactuated flexure-based grippers, aimed at maximizing the range of graspable circular objects under a given load. This method achieves a superior design faster than state-of-the-art methods that optimize all design parameters simultaneously. A prototype constructed using rapid-prototyping validates the usage of the design method, and experimentally illustrates gripper performance

Heptameric association of light-harvesting complex II trimers in partially solubilized photosystem II membranes

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of seven trimeric light-harvesting complex II proteins, The complex was readily observed in partially-solubilized Tris-washed photosystem II membranes from spinach but was also found to occur, with a low frequency, in oxygen-evolving photosystem II membranes. The structure reveals sis peripheral trimers with the same rotational orientation and a central trimer with the opposite orientation. We conclude that the heptamer represents a naturally occurring aggregation state of part of the light-harvesting complex II trimers in the thylakoid membranes. (C) 1999 Federation of European Biochemical Societies.</p

Supporting measurements or more averages? How to quantify cerebral blood flow most reliably in 5 minutes by arterial spin labeling

Purpose To determine whether sacrificing part of the scan time of pseudo-continuous arterial spin labeling (PCASL) for measurement of the labeling efficiency and blood T1 is beneficial in terms of CBF quantification reliability. Methods In a simulation framework, 5-minute scan protocols with different scan time divisions between PCASL data acquisition and supporting measurements were evaluated in terms of CBF estimation variability across both noise and ground truth parameter realizations taken from the general population distribution. The entire simulation experiment was repeated for a single-post-labeling delay (PLD), multi-PLD, and free-lunch time-encoded (te-FL) PCASL acquisition strategy. Furthermore, a real data study was designed for preliminary validation. Results For the considered population statistics, measuring the labeling efficiency and the blood T1 proved beneficial in terms of CBF estimation variability for any distribution of the 5-minute scan time compared to only acquiring ASL data. Compared to single-PLD PCASL without support measurements as recommended in the consensus statement, a 26%, 33%, and 42% reduction in relative CBF estimation variability was found for optimal combinations of supporting measurements with single-PLD, free-lunch, and multi-PLD PCASL data acquisition, respectively. The benefit of taking the individual variation of blood T1 into account was also demonstrated in the real data experiment. Conclusions Spending time to measure the labeling efficiency and the blood T1 instead of acquiring more averages of the PCASL data proves to be advisable for robust CBF quantification in the general population