High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media

Large two-dimensional crystals of the light-harvesting chlorophyll a/b-protein complex (LHC-II) from the photosynthetic membrane of pea chloroplasts were grown by a new method from detergent solution. The structure of these crystals was examined by electron crystallography, using three different media to preserve high-resolution detail: vitrified water, glucose and tannin. The crystals diffracted electrons to at least 3.2 A resolution in all three media. R-factors between the three data sets of electron diffraction amplitudes ranged from 6.4% to 14.3%. Fourier difference maps were generated and compared to a projection map of the complex at 3.4 A resolution. No significant differences were found, proving that all three media preserved the native structure of LHC-II at high resolution. The probability of recording high-quality electron diffraction patterns with tannin was 90%. With glucose and water this probability was lower by a factor of 10 to 20, suggesting that tannin may be preferable as a preserving medium for sensitive biological specimens.     

The resolution and biochemical characterization of subcomplexes of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II)

LHC II isolated from carnation leaves has been solubilized and resolved by a newly developed, vertical-bed non-denaturing isoelectric focusing in polyacrylamide slab gels to yield three trimeric subcomplexes focusing at pH 4.52, 4.42 and 4.37 (designated a, b and c, respectively), comprising approximately 38%, 24% and 38% of the chlorophyll. The spectroscopic data demonstrated a close similarity among LHC II subcomplexes concerning their chlorophyll content and organization. The most alkaline and the most acidic subcomplex contained the 27 kDa polypeptide of LHC II while the intermediate pI fraction contained both LHC II polypeptides, i.e. 27 kDa and 26 kDa ones associated at 2:1 stoichiometry. The 27 kDa polypeptide could be resolved by denaturing isoelectrofocusing into 10 pI molecular isoforms covering 5.90–4.20 pH range. Three of the isoforms were found in the subcomplexes a and b and eight in the subcomplex c. The 26 kDa polypeptide comprised the unique pI molecular isoform focusing at pH 5.61.Abbreviations CBB G-250 Coomassie Brilliant Blue G-250 - chl chlorophyll - DM n-dodecyl--d-maltoside - EDTA ethylendiaminotetraacetic acid - IEF isoelectric focusing - LHC II the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - LHCP II apoprotein of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - NP-40 polyethyleneglycol-p-isooctylphenyl ether - pI isoelectric point - OG octyl--d-glucopyranoside - PS II Photosystem II - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TCA trichlorooacetic acid     

Evidence for an O2-barrier in the light-harvesting chlorophyll-a/b-protein complex LHC II

An O₂-barrier in the intact light-harvesting complex LHC II protects chlorophylls (Chl) and xanthophylls (Car) from photooxidation. Direct evidence for the limited access of O₂ to pigment sites comes from the decay kinetics of the first excited triplet state of Car (³Car-). The LHC-bound ³Car- in air-saturated solution decays mono-exponentially with a lifetime of 6.7-7.1 µs as compared to the approx. 1.2 µs of the -carotene triplet in hexane and the 8.8-9 µs observed for both systems under anaerobiosis. Further properties of the photostable complex are the limited access of protons to pigment sites and the efficient energy transfer from ¹Car- to Chl-a and from ³Chl- to Car. Fatty acids with increasing chain length increasingly lower both, the efficiency of the O₂ barrier and the photo- and acid stability of the LHC-bound pigments while singlet and triplet energy transfer between the pigments is maintained. Therefore, the close proximity of Chl and Car is not sufficient to protect the pigments from photooxidation; in addition, an O₂-barrier limiting the access of O₂ to pigment sites is required for efficient photoprotection. Structural properties of the photostable LHC II possibly underlying its O₂-barrier function are discussed.     

Identification of Lhcb1/Lhcb2/Lhcb3 heterotrimers of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II)

Using non-denaturing isoelectric focusing in polyacrylamide vertical slab gel, we have purified to homogeneity three trimeric subcomplexes of LHC II from Arabidopsis thylakoid membranes. The polypeptide composition of the subcomplexes were studied by immunoblotting. Our results indicate the existence in vivo of LHC II heterotrimers containing Lhcb1, Lhcb2 and Lhcb3 gene products.     

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.     

Time-Resolved fluorescence studies on the internal motion of chlorophyll a of light-harvesting chlorophyll a/b-protein complex in lipid membranes

By analyzing the steady state and time-resolved fluorescence anisotropy, the internal motions of chlorophyll a of light-harvesting chlorophyll a/b-protein complex (LHCII) were characterized in a dimyristoylphosphatidylcholine (DMPC) liposome. Corresponding to the thermotropic phase of the membrane, chlorophyll a showed an unique internal motion in LHCII. At the gel phase, two motional components, one fast and the other slow, were observed, which would originate in the heterogeneity of the mutual orientation and the binding site of the chlorophyll a in LHCII. Interestingly, the faster motion was suppressed and only the slower segmental rotation with the larger motional amplitude was allowed on the phase transition to a liquid crystalline phase.     

Resolution of the light-harvesting chlorophyll a/b-protein of vicia faba chloroplasts into two different chlorophyll-protein complexes.

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll a/b-protein was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll a/b ratio of 0.9 and is associated with polypetides of 24,000 and 23,000 daltons. The 24,000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23,000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll a/b ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23,000. The two 23,000 dalton polypeptides occurring in both complexes are not identical.     

Monoclonal antibodies produced against antigenic determinants present in complex mixtures of proteins

This overview provides information concerning the production of monoclonal antibodies (MAbs) against specific antigenic determinants present in complex mixtures of proteins. We review five specific techniques for the production of these antibodies (Abs): (a) So-called "shotgun," non-selective approach; (b) cascade procedure; (c) lymphocyte "panning"; (d) cyclophosphamide elimination of unwanted Ab producers; and finally (e) use of polyclonal antisera to extinguish unwanted antibody production. We discuss the relative advantages and disadvantages of these various procedures, and suggest alternative strategies by which specific MAbs might be generated.     

Preferential accumulation of apoproteins of the light-harvesting chlorophyll a/b-protein complex in greening barley leaves treated with 5-aminolevulinic acid

In order to study the coordinate accumulation of chlorophyll (Chl) and apoproteins of Chl-protein complexes (CPs) during chloroplast development, we examined changes in the accumulation of the apoproteins in barley (Hordeum vulgare L.) leaves when the rate of Chl synthesis was altered by feeding 5-aminolevulinic acid (ALA), a precursor of Chl biosynthesis. Pretreatment with ALA increased the accumulation of Chl a and Chl b 1.5- and 2.3-fold, respectively, after 12 cycles of intermittent light (2 min light followed by 28 min darkness). Apoproteins of the light-harvesting Chl a/b-protein complex of photosystem II (LHCII) were increased 2.4-fold with ALA treatment. However, apoproteins of the P700-Chl a-protein complex (CP1) and the 43-kDa apoprotein of a Chl a-protein complex of photosystem II (CPa) were not increased by ALA application. With respect to CPs themselves, LHCII was increased when Chl synthesis was raised by ALA feeding, whereas CP1 exhibited no remarkable increase. These results indicate that LHCII serves a role in maintaining the stoichiometry of Chl to apoproteins by acting as a temporary pool for Chl molecules.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll - CP chlorophyll-protein complex - CPa chlorophyll a-protein complex of PSII - CP1 P700-chlorophyll a-protein complex - LDS lithium dodecyl sulfate - LHCII light-harvesting chlorophyll a/b-protein complex of PSII This work was supported by the Grants-in-Aid for Scientific Research (04304004) from the Ministry of Education, Science and Culture, Japan.     

Isolation of two different subpopulations of the light-harvesting chlorophyll a/b complex of photosystem II (LHCII)

Isolated LHCII from spinach has been solubilized and fractionated by non-denaturing isoelectric focusing to yield two subpopulations with different polypeptide but equal chlorophyll composition. One LHCII subpopulation contains only a 27 kDa polypeptide while the other contains the 27 and 25 kDa polypeptides in about equal amounts. The polypeptide patterns of the two subpopulations closely correspond to those suggested previously for the inner LHCII and peripheral LHCII, respectively.     

Role of spermidine in the stabilization of the apoprotein of the light-harvesting chlorophyll a/b-protein complex of photosystem II during leaf senescence process

Barley leaf discs maintained in dark accumulated a massive amount of putrescine (Put), lost chlorophyll and senescenced rapidly. At the same time RNase activity increased significantly. Exogenous spermidine (Spd) inhibited RNase activity, the loss of chlorophyll and degradation of the proteins from thylakoid membranes. Using SDS-PAGE and immunoblot analysis it was shown that spermidine was effective in the retardation of the loss of LHCPII observed in water-treated detached leaves. Analysis of PSII particles isolated from leaf fragments floated in water in the dark revealed the presence of Put, Spd and Spm. In spermidine treated leaves the level of this polyamine in photosystem II was above 5-fold higher than in control. The experimental findings obtained in this study provide evidence that applied spermidine interacts directly with thylakoid membranes so that they become more stable to degradation during senescence.     

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12–58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E⁶⁵VIHARWAM⁷³ led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159–166] is proposed.Abbreviations BSA bovine serum albumin faction V - ELIPs early light-inducible proteins - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LHCP light-harvesting chlorophyll a/b-protein - LHC IIb light-harvesting complex associated with Photosystem II - pLHCP precursor to LHCP - Rubisco ribulose 1,5-biphosphate carboxylase-oxygenase - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis     

Properties of light-harvesting chlorophyll a/b-protein, and photosystem I chlorophyll a-protein, purified from digitonin extracts of spinach chloroplasts by isoelectrofocusing

A procedure for purifying both light-harvesting chlorophylla/b-protein and photosystem I chlorophyll -protein from digitoninextracts of spinach chloroplasts is described. This procedureuses isoelectrofocusing on Ampholine at the last step and permitsisolating all of the chlorophyll-proteins from the same extractin a better yield and a highly pure state. The purified light-harvesting chlorophyll a/b-protein whichhas an isoelectric point (pi) of 4.35 (?0.1) and a single polypeptideof 24 kilodaltons (kD), shows slightly higher chlorophyll a/Aratio of 1.35 than the values reported for the preparationsobtained by anionic detergents. This chlorophyll-protein exhibitsa markedly high and sharp fluorescence band at 681 nm at 77?Kwhich is not found on the chloroplast emission spectrum. Photosystem I chlorophyll a-protein focuses on Ampholine intotwo bands with pi values of 4.75 (?0.1) and 4.80 (?0.1). Thesetwo fractions show the same absorption spectra (maximum at 678nm at room temperature) and emission spectra (maximum at 734nm at 77?K) and have the same constituent polypeptides: onelarge band at 55–64 kD and six minor bands (21.5, 20,19, 18, 16 and 15 kD). The polypeptide composition and the P-700to chlorophyll a ratio (1 to ca. 80) of this preparation arevery similar to those of the photosystem I reaction center preparationobtained from Swiss chard chloroplasts by Bengis and Nelson(8). (Received October 31, 1978; )     

Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.     

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

A cytochrome b ₆ f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a ‘high light’ acclimation state. The cytochrome b ₆ f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Characterization of three forms of light-harvesting chlorophyll a/b-protein complexes of photosystem II isolated from the green alga, Dunaliella salina

Three forms of light-harvesting chlorophyll a/b-protein complexes of photosystem II (LHC II) were isolated from the thylakoid membranes of Dunaliella salina grown under different irradiance conditions. Cells grown under a low intensity light condition (80 micromol quanta m(-2) s(-1)) contained one form of LHC II, LHC-L. Two other forms of LHC II, LHC-H1 and LHC-H2, were separated from the cells grown under a high intensity light condition (1,500 micromol quanta m(-2) s(-1)). LHC-L and LHC-H1 showed an apparent particle size of 310 kDa and contained four polypeptides of 31, 30, 29 and 28 kDa. LHC-H2, with a particle size of 110 kDa, consisted of 30 and 28 kDa polypeptides. LHC-L contained 7.5 molecules of Chl a, 3.2 of Chl b and 2.1 of lutein per polypeptide, analogous to the content in higher plants. LHC-H1, with 5.6 molecules of Chl a, 2.5 of Chl b and 1.8 of lutein per polypeptide was similar to that in the green alga Bryopsis maxima. LHC-L and LHC-H1 maintained high efficiency energy transfer from Chl b and lutein to Chl a molecules. LHC-H2 showed a high Chl a/b ratio of 7.5 and contained 3.4 molecules of Chl a, 0.5 of Chl b and 1.4 of lutein per polypeptide. Chl b and lutein could not completely transfer the excitation energy to Chl a in LHC-H2.