Crystallization of the light-harvesting chlorophyll a/b complex within thylakoid membranes.

We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.

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Studies on the Herbicide Binding Site in Isolated Photosystem II Core Complexes from a Flat-Bed Isoelectrofocusing Method

Zeitschrift für Naturforschung C ◽

10.1515/znc-1990-0510 ◽

1990 ◽

Vol 45 (5) ◽

pp. 366-372 ◽

Cited By ~ 6

Author(s):

M. T. Giardi ◽

J. Barber ◽

M. C. Giardina ◽

R. Bassi

Keyword(s):

Light Harvesting ◽

Resistant Mutant ◽

Separation Procedure ◽

Chlorophyll B ◽

Core Complex ◽

Light Harvesting Complexes ◽

Lhc Ii ◽

Ps Ii ◽

Α Subunit ◽

Ps Ii Core

Abstract Isoelectrofocusing has been used to separate various chlorophyll-protein complexes of photosystem two (PS II). Light-harvesting complexes containing chlorophyll a and chlorophyll b (LHC II) were located in bands having p/s in the region of 4.5. At slightly higher pH other light-harvesting complexes containing little or no chlorophyll b were found. In the most basic region of the isoelectrofocusing gel, were located PS II core complexes characterized by containing the proteins of CP47, CP43, D 1, D 2 and α-subunit of cytochrome b559. The number of PS II core bands depended on the particular conditions employed for the separation procedure and in some cases were contaminated by CP 29. It is suggested that this heterogeneity resulting from different protonation states of the PS II. The least-acidic PS II core complex (pI 5.5) was found to bind the herbicides atrazine, diuron and dinoseb. In contrast, a PS II core complex with a p / of 4.9 bound only diuron. Its inability to bind atrazine was shown to be due to the low pH but no such explanation could be found for dinoseb. When atrazine-resistant mutant Senecio vulgaris was used, no binding of radioactive atra zine was observed with the PS II cores having a p i of 5.5. It is therefore suggested that the normal atrazine binding observed with PS II cores involves the high affinity site detected with intact membranes. With the PS II cores, however, this site has a reduced affinity probably due to structural modification in the D 1-polypeptide resulting from the isolation procedures.

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Inhibition of State Transition and Light-Harvesting Complex II Phosphorylation-Mediated Changes in Excitation Energy Distribution in the Thylakoids of SANDOZ 9785-Treated Plants

Zeitschrift für Naturforschung C ◽

10.1515/znc-1995-1-212 ◽

1995 ◽

Vol 50 (1-2) ◽

pp. 77-85

Author(s):

Manoj K. Joshi ◽

Prasanna Mohanty ◽

Salil Bose

Keyword(s):

Energy Distribution ◽

State Transition ◽

Light Harvesting ◽

Lhc Ii ◽

Ps Ii ◽

Light Harvesting Complex ◽

Antenna Size ◽

Complex Ii ◽

Ps I ◽

Light Harvesting Complex Ii

Abstract Thylakoids isolated from SAN 9785 (4-chloro-5-dimethylamino-2-phenyl-3(2H)-pyridazi-none)-treated pea plants showed an inhibition of “state transition” and the light-harvesting complex II (LHC II) phosphorylation-mediated changes in the energy distribution between photosystem II (PS II) and photosystem I (PS I) as measured by a decrease in PS II and an increase in PS I fluorescence yield. Interestingly, in these thylakoids the extent of phosphorylation-induced migration of light-harvesting complex (LHC II-P) to non-appressed membrane regions was only marginally inhibited. We propose that the suppression in the ability for “state transition” by SANDOZ 9785 (SAN 9785) treatment occurs due to a lack of effective coupling of the migrated LHC II-P and PS I. Since we observed a decrease in the antenna size of PS I of the treated plants, the lack of effective coupling is attributed to this decrease in the antenna size of PS I.

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Acclimation of the Photosynthetic Apparatus to Photosystem I or Photosystem II Light: Evidence from Quantum Yield Measurements and Fluorescence Spectroscopy of Cyanobacterial Cells

Zeitschrift für Naturforschung C ◽

10.1515/znc-1989-1-219 ◽

1989 ◽

Vol 44 (1-2) ◽

pp. 109-118 ◽

Cited By ~ 32

Author(s):

Anastasios Melis ◽

Conrad W. Mullineaux ◽

John F. Allen

Keyword(s):

Quantum Yield ◽

Photosystem Ii ◽

Excitation Energy ◽

Oxygen Evolution ◽

Photosystem I ◽

Light Harvesting ◽

Ps Ii ◽

Ps I ◽

Synechococcus 6301 ◽

Light Harvesting Antenna

Abstract Cells of the cyanobacterium Synechococcus 6301 were grown under illumination whose spectral composition favoured absorption either by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II) or by the chlorophyll (Chi) a light-harvesting antenna of photosystem I (PS I). Cells grown under PS I-light developed relatively high PS II/PS I and PBS/Chl ratios. Cells grown under PS II-light developed relatively low PS II/PS I and PBS/Chl ratios. Thus, the primary difference between cells in the two acclimation states appeared to be the relative concentration of PBS-PS II and PS I complexes in the thylakoid membrane. Measurements of the quantum yield of oxygen evolution suggested a higher efficiency of cellular photosynthesis upon the adjustment of photosystem stoichiometry to a specific light condition. The quantum yield of oxygen evolution was nevertheless lower under PBS than Chi excitation, suggesting quenching of excitation energy in the photochemical apparatus of PS II in Synechococcus 6301. This phenomenon was more pronounced in the PS II-light than in the PS I-light grown cells. Room temperature and 77 K fluorescence emission spectroscopy indicated that excess excitation energy in the PBS was not transferred to PS I, suggesting the operation of a non-radiative and non-photochemical decay of excitation energy at the PBS-PS II complex. This non-photochemical quenching was specific to conditions where excitation of PS II occurred in excess of its capacity for useful photochemistry.

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Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen.

The Journal of Cell Biology ◽

10.1083/jcb.115.2.321 ◽

1991 ◽

Vol 115 (2) ◽

pp. 321-328 ◽

Cited By ~ 46

Author(s):

W F Ettinger ◽

S M Theg

Keyword(s):

Oxygen Evolving Complex ◽

Ps Ii ◽

Thylakoid Lumen ◽

Core Proteins ◽

Before And After ◽

Detergent Treatment ◽

Extrinsic Proteins ◽

Oxygen Evolving ◽

Triton X ◽

Ps Ii Core

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.

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Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

Molecular Biology of the Cell ◽

10.1091/mbc.6.7.929 ◽

1995 ◽

Vol 6 (7) ◽

pp. 929-944 ◽

Cited By ~ 227

Author(s):

S Mayor ◽

F R Maxfield

Keyword(s):

Cell Surface ◽

Tyrosine Kinases ◽

Plasma Membranes ◽

Cytoplasmic Proteins ◽

Nonionic Detergent ◽

Detergent Treatment ◽

Carboxy Terminal ◽

Almost All ◽

Membrane Anchoring ◽

Triton X

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined.

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An e.x.a.f.s. study of the manganese O2-evolving complex in purified Photosystem II membrane fractions. The S1 and S2 states

Biochemical Journal ◽

10.1042/bj2850569 ◽

1992 ◽

Vol 285 (2) ◽

pp. 569-576 ◽

Cited By ~ 46

Author(s):

D J MacLachlan ◽

B J Hallahan ◽

S V Ruffle ◽

J H A Nugent ◽

M C W Evans ◽

...

Keyword(s):

Photosystem Ii ◽

Light Harvesting ◽

Manganese Complex ◽

X Ray ◽

Bond Lengths ◽

Detergent Treatment ◽

S2 State ◽

Triton X ◽

Oxygen Bond

Manganese K-edge X-ray spectra have been obtained for Photosystem II samples isolated using Triton X-100 detergent and samples further purified by n-heptyl beta-D-thioglucoside detergent treatment to remove light-harvesting polypeptides and low-affinity calcium. The structure of the manganese complex is very similar for the two detergent preparations used. Analysis of the e.x.a.f.s. spectra for samples in the S1 and S2 states indicate changes in bond lengths for the shells of oxygen/nitrogen atoms. For the S1 state, oxygen shells at 0.181 and 0.193 nm (1.81 and 1.93 A) were observed and one manganese shell at 0.270 nm (2.70A). In the S2 state the oxygen bond lengths are longer at 0.184 and 0.200 nm (1.84 and 2.00 A). Additionally a shell of scatterers at 0.37 nm (3.7 A) was observed in both states which could be fitted to models with calcium scatterers at this distance.

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High-Resolution electron crystallography of the light-harvesting chlorophyll-a/b protein complex from chloroplast membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181816 ◽

1990 ◽

Vol 48 (1) ◽

pp. 610-610

Author(s):

Werner Kühlbrandt ◽

Da Neng Wang ◽

K.H. Downing

Keyword(s):

High Resolution ◽

Electron Diffraction ◽

Chlorophyll A ◽

Protein Complex ◽

Structural Integrity ◽

Light Harvesting ◽

Lhc Ii ◽

Diffraction Patterns ◽

Difference Fourier ◽

2D Crystals

The light-harvesting chlorophyll-a/b protein complex (LHC-II) is the most abundant membrane protein in the chloroplasts of green plants where it functions as a molecular antenna of solar energy for photosynthesis. We have grown two-dimensional (2d) crystals of the purified, detergent-solubilized LHC-II . The crystals which measured 5 to 10 μm in diameter were stabilized for electron microscopy by washing with a 0.5% solution of tannin. Electron diffraction patterns of untilted 2d crystals cooled to 130 K showed sharp spots to 3.1 Å resolution. Spot-scan images of 2d crystals were recorded at 160 K with the Berkeley microscope . Images of untilted crystals were processed, using the unbending procedure by Henderson et al . A projection map of the complex at 3.7Å resolution was generated from electron diffraction amplitudes and high-resolution phases obtained by image processing .A difference Fourier analysis with the same image phases and electron diffraction amplitudes recorded of frozen, hydrated specimens showed no significant differences in the 3.7Å projection map. Our tannin treatment therefore does not affect the structural integrity of the complex.

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