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• W2006823377 abstract "The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refoldedin vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Qy transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem. The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refoldedin vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Qy transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem. chlorophyll chlorophyll protein light-harvesting complex of photosystem II high pressure liquid chromatography wild type In green plants light energy for photosynthesis is collected by an antenna system, made of many homologous proteins belonging to theLhc multigene family (1Green B.R. Pichersky E. Kloppstech K. Trends Biochem. Sci. 1991; 16: 181-186Abstract Full Text PDF PubMed Scopus (234) Google Scholar). These pigment proteins are organized around photosynthetic reaction centers to form supramolecular complexes embedded into the thylakoid membranes. Lhc proteins bind about 70% of the pigments involved in plant photosynthesis. Understanding of energy transfer processes in the antenna and reaction centers requires recognition of the topological organization of subunits (2Bassi R. Sandonà D. Croce R. Physiol. Plant. 1997; 100: 769-779Crossref Google Scholar, 3Boekema E.J. Hankamer B. Bald D. Kruip J. Nield J. Boonstra A.F. Barber J. Rögner M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 175-179Crossref PubMed Scopus (273) Google Scholar, 4Harrer R. Bassi R. Testi M.G. Schäfer C. Eur. J. Biochem. 1998; 255: 196-205Crossref PubMed Scopus (68) Google Scholar) and knowledge of three major parameters, namely: (i) the distances between chromophores; (ii) the mutual orientation of dipole transition moments; and (iii) the absorption/fluorescence energy levels. Although the elucidation of LHCII structure at 3.4 Å resolution (5Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1542) Google Scholar) has allowed localization of chlorophyll-binding sites and of their relative distances, identification of transition dipole orientation and energy levels are precluded by insufficient resolution of the structure so far obtained or are not accessible by structural methods. Among Lhc proteins the most abundant is LHCII, which can be isolated as an heterotrimeric complex of the Lhcb1–3 gene products (6De Luca C. Varotto C. Svendsen I. Polverino-De Laureto P. Bassi R. J. Photochem. Photobiol. 1998; 49: 50-60Crossref Scopus (7) Google Scholar). LHCII binds 7 Chl1 a, 5 Chl b, and three xanthophyll molecules/mol of polypeptide (1.6–1.8 mol of lutein, 0.2–0.4 mol of violaxanthin, and 1.0 mol of neoxanthin) and is the best characterized Lhc polypeptide. (5Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1542) Google Scholar, 7Dainese P. Bassi R. J. Biol. Chem. 1991; 266: 8136-8142Abstract Full Text PDF PubMed Google Scholar, 8Bassi R. Pineau B. Dainese P. Marquardt J. Eur. J. Biochem. 1993; 212: 297-303Crossref PubMed Scopus (350) Google Scholar, 9Connelly J.P. Muller M. Bassi R. Croce R. Holzwarth A.R. Biochemistry. 1997; 36: 281-287Crossref PubMed Scopus (116) Google Scholar, 10Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Knowledge of the energy transfer factors for this protein would be a major step toward elucidation of light harvesting function. In this study we have used mutation analysis with the aim of the identification and characterization of the chromophores bound to each site; a series of mutant apoproteins was constructed by overexpression in bacteria of theLhcb1 gene in which individual chlorophyll-binding residues (5Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1542) Google Scholar) were substituted for by residues unable to coordinate porphyrins. Upon in vitro refolding with purified pigments, proteins missing specific chromophores were obtained in their monomeric form, which could be trimerized by addition of lipids (11Hobe S. Prytulla S. Kühlbrandt W. Paulsen H. EMBO J. 1994; 13: 3423-3429Crossref PubMed Scopus (156) Google Scholar). In this work we focus on the monomeric form of LHCII to avoid the effect of inter-subunit interactions on the spectral properties of individual chlorophylls that could complicate the attribution of spectral forms to individual sites. Biochemical analysis and differential absorption spectroscopy allows a proposed map for the chemical nature and the absorption properties of chlorophylls within individual sites. Plasmids were constructed using standard molecular cloning procedures (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Bacterial hosts wereEscherichia coli TG1 strain (13Straus S.D. Fells G.A. Wewers M.D. Courtney M. Tessier L.H. Tolstoshev P. Lecocq J.P. Crystal R.G. Biochem. Biophys. Res. Commun. 1985; 130: 1177-1184Crossref PubMed Scopus (29) Google Scholar) and SG13009 strain (14Gottesman S. Halpern E. Trisler P. J. Bacteriol. 1981; 148: 265-273Crossref PubMed Google Scholar). cDNA of lhcb1 from Zea mays was a kind gift of Dr. Matsouka (15Matsouka M. Kano-Murakami Y. Yamamoto N. Nucleic Acids Res. 1987; 15: 6302Crossref PubMed Scopus (28) Google Scholar). Mutations were obtained according to the method of Yukenberg et al. (16Yukenberg P.D. Withey F. Geisselsoder J. McClary J. McPherson M.J. Directed Mutagenesis: A Practical Approach. IRL Press, Oxford1991: 27-48Google Scholar). The sequence was determined by the dideoxy method (17Sanger F. Nicken S. Carlson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5468Crossref PubMed Scopus (52769) Google Scholar) by an automated apparatus (Applied Biosystems model 377). LHCII was isolated from the SG13009 strain transformed with the lhcb1 construct following a protocol previously described (10Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 18Nagai K. Thogersen H.C. Methods Enzymol. 1987; 153: 461-481Crossref PubMed Scopus (347) Google Scholar, 19Paulsen H. Rümler U. Rüdiger W. Planta. 1990; 181: 204-211Crossref PubMed Scopus (227) Google Scholar). Purification was performed as described in Giuffra et al. (20Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar) with the modifications reported in Croce et al. (10Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Was performed by ion exchange chromatography (20Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar). For determination of pigment to protein stoichiometry, it was necessary to obtain fully purified complexes that did not contain any residual contamination by bacterial proteins. The reconstituted LHCII was thus purified by preparative isoelectrofocusing (21Dainese P. Hoyer-Hansen G. Bassi R. Photochem. Photobiol. 1990; 51: 693-703Crossref PubMed Google Scholar) followed by ultracentrifugation in glycerol gradient (15–40% including 0.06% dodecylmaltoside and 10 mm Hepes, pH 7.6; run was for 12 h at 60,000 rpm in SW60 Beckman rotor) to eliminate ampholytes. The concentration of the LHCII apoprotein purified from E. coli inclusion bodies was determined by the bicinchoninic acid assay (22Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). For stoichiometric (pigments/protein ratio) determination, the protein concentration was determined by the ninhydrin method (23Hirs C.H.W. Methods Enzymol. 1967; 11: 325-329Crossref Scopus (208) Google Scholar). Chlorophyll concentration was determined by the method of Porra et al. (24Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4743) Google Scholar). HPLC analysis was as in Ref. 25Gilmore A.M. Yamamoto H.Y. J. Chromatogr. 1991; 543: 137-145Crossref Scopus (412) Google Scholar. Chlorophyll to carotenoid ratio and Chl a/b ratio was independently measured by fitting of acetone extract spectra with the spectra of individual purified pigments as described previously (9Connelly J.P. Muller M. Bassi R. Croce R. Holzwarth A.R. Biochemistry. 1997; 36: 281-287Crossref PubMed Scopus (116) Google Scholar). Absorption spectra were obtained using a SLM-Aminco DW-2000 spectrophotometer at room temperature. Fluorescence excitation and emission spectra were obtained by using a Jasco-600 spectrofluorimeter. CD spectra were obtained at 8 °C with a Jasco FP-777 spectropolarimeter. Samples were in 10 mm Hepes, pH 7.6, 0.06% dodecylmaltoside, 20% glycerol. Chlorophyll concentration was about 10 g/ml for CD and absorption measurements and 0.01 g/ml for fluorescence measurements. Analysis of spectra by Gaussian deconvolution and second derivative analysis was performed by using the (OriginTM MicroCal. Soft. Inc.) software package. Mutations were performed on the nine amino acid residues in the LHCII sequence that have been proposed from the structure to provide ligation of Chl molecules (5Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1542) Google Scholar). In the case of Chl A6 ligand proposed to be the peptidyl carbonyl group of glycine 78, which is made available for coordination by being not involved in intra-helix H-bonds with the α-amino group of the proline residue 82, we have changed proline 82 into valine to eliminate the peptidyl-carbonyl of Gly78from coordination by H binding to Val82. In the case of ligation by glutamate/arginine ionic pairs, mutation of the glutamate only would leave a noncompensated charged residue into the hydrophobic core of the protein thus disturbing protein folding (26Bassi R. Croce R. Cugini D. Sandonà D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10056-10061Crossref PubMed Scopus (191) Google Scholar); we therefore performed double mutations of both glutamate and arginine to noncharged residues in the case of sites A1 and A4. In the case of site B5 both the single mutants and the double mutants were constructed and analyzed. All mutations yielded inclusion bodies with a yield of 3.5 ± 32 mg/g of bacterial cells. TableI gives a summary of mutant proteins analyzed in this work.Table ILHCII mutantsA1, E180V/R70IA2, N183VA3, Q197VA4, E65V/R185LA5, H68IA6, P82VB3, H212VB5.1, E139LB5.2, F139L/R142LB6.1, Q131VB6.2, Q131EEach mutant is designated by the site as assigned by Kuhlbrandt and co-workers (11Hobe S. Prytulla S. Kühlbrandt W. Paulsen H. EMBO J. 1994; 13: 3423-3429Crossref PubMed Scopus (156) Google Scholar). The targeted amino acid residues and their substitutions are shown. Open table in a new tab Each mutant is designated by the site as assigned by Kuhlbrandt and co-workers (11Hobe S. Prytulla S. Kühlbrandt W. Paulsen H. EMBO J. 1994; 13: 3423-3429Crossref PubMed Scopus (156) Google Scholar). The targeted amino acid residues and their substitutions are shown. To detect differences in pigment binding induced by mutations that could be attributed to the native protein in thylakoid membrane, it was essential to reproduce the properties of LHCII by in vitroreconstitution of recombinant Lhcb1 apoprotein. In preliminary experiments LHCII was reconstituted by using different Chl a/Chl b/xanthophyll ratios into the reconstituted mixture. The pigment to apoprotein ratio was also explored. LHCII purified from maize thylakoids binds 7 molecules of Chl a, 5 Chl b, 1.8 lutein, 0.2 violaxanthin, and 1.0 neoxanthin per polypeptide. This composition was reproduced with Lhcb1 very closely (7 Chl a, 5 Chl b, 1.68 lutein, 0.32 violaxanthin, and 1.0 neoxanthin) by using pigment mixture during reconstitution with Chl a/b ratio of 2.3 and Chl a+b/carotenoids = 4.2. The composition of the carotenoid extract was the following: neoxanthin/violaxanthin/lutein/carotene = 16/16/50/18. The ratio between protein and Chl in the mixture was 0.57 (w/w). At first we encountered difficulty in reproducing this result routinely because of fluctuations in the a/b ratio of the reconstituted complex between 1.6 and 1.4. This was due to partial aggregation of pigments during the procedure of refolding, thus yielding a green band close to the bottom of the sucrose gradient and causing random changes in the Chl a/b ratio of the pigments actually available for binding to the protein. Decreasing pigment concentration in the reconstitution mixture avoided aggregation. However, exceedingly lower pigment concentration led to a LHCII protein binding 11 Chl rather than 12 (one Chl b was missing). The conditions used in this work allowed reconstitution of LHCII binding 12 Chl with Chl a/b ratio of 1.4 in 75% of the experiments. In each experiment one WT sample was included and the products of experiments in which the Chl a/b ratio of WT LHCII differed from 1.4 by more than 0.03 were discarded. The data reported here refer to three independent reconstitution experiments for each mutant protein. WT LHCII was reconstituted with a yield of 35% on a protein basis (Fig.1). Mutant proteins were reconstituted with a similar yield with some exceptions; the A1 mutant (E180L/R70I) had a yield of 10%, suggesting that this ionic pair is important in stabilizing the structure, whereas, interestingly, mutants in the other intra-helix ionic pair (A4 site, E65V/R185L) did not show a decrease in reconstitution yield. Three mutations in helix C, disrupting the intra-helix ionic pair (B5.1 and B5.2) or the Chl binding (B6.1) decreased the reconstitution yield to 25, 12, and 25%, respectively. Stability of the complexes was checked by keeping the samples in ice or at room temperature and repeating absorption spectra during the following 24 h. WT and all mutants but A1 showed no shift in the wavelength of the Qy transition peak within 0.2 nm for 24 h in ice or 6 h at RT. The A1 mutant was stable for 4–6 h in ice. The function of LHCII is the light harvesting by the three different chromophore types present in the complex and excitation energy transfer to Chl a prior to further transfer to photosynthetic reaction centers. High efficiency of energy transfer can only be obtained if the relative distances and orientation between chromophores is maintained. This can be assessed by fluorescence emission analysis using three different wavelengths of excitation (440, 475, and 500 nm) specifically absorbed by Chl a, Chl b, and xanthophylls. Emission from impaired individual or groups of chlorophyll will show up at different wavelengths. For WT LHCII and most of the mutant proteins, fluorescence emission spectra typically showed a single major peak at 681 nm. The spectra were essentially identical irrespective of excitation wavelength, implying energy transfer and equilibration between all the bound pigments (Fig.2). This indicates that mutations did not significantly disrupt protein structure or disturb pigment-pigment interactions between chromophores not specifically affected by the mutation. Nevertheless, small differences in peak emission wavelengths indicate that the relative distribution of the Chl absorption forms was modified. Some Chl b emission at 660 nm was observed in the helix C mutants B5.1, B5.2, and B6.1, indicating that a fraction of the energy absorbed by Chl b could not be transferred to Chl a. However, even in the case of B5.1 and B6, most of energy was equilibrated as shown by the identical shape of the spectra above 670 nm. The only clear exception to this pattern was the B5.2 mutant, in which energy from carotenoid excitation at 500 nm was transferred to a different Chl a pool emitting at higher wavelengths with respect to the overall Chl a emission excited at 440 nm. This implies that energy transfer between two different Chl a groups was impaired. The A2 mutant was equilibrated, but its emission peak was blue shifted by 4 nm, indicating the Chl affected by the mutation is probably the chromophore with the lowest energy level in the complex. Site A2 was suggested to be important for energy transfer toward neighboring antenna proteins on the basis of its protruding position in LHCII structure (5Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1542) Google Scholar). To verify whether mutations have affected selectively different Chl absorption forms, we have analyzed WT and mutant LHCII proteins by room temperature absorption spectroscopy. The Qy region of the spectra is shown in Fig.3 with their second derivative analysis. WT monomeric recombinant LHCII shows two broad peaks respectively at 652 and 674 nm (Fig. 3) as previously shown for the native complex purified from thylakoids (27Bassi R. Silvestri M. Dainese P. Giacometti G.M. Moya I. J. Photochem. Photobiol. 1991; 6: 381-394Google Scholar, 28Peterman E.J.G. Dukker F.M. van Grondelle R. Van Amerongen H. Biophys. J. 1995; 69: 2670-2678Abstract Full Text PDF PubMed Scopus (158) Google Scholar). This protein could be made into trimers by incubation with lipid extract leading to the changes on absorption and CD spectra previously reported to be due to trimerization in native LHCII (27Bassi R. Silvestri M. Dainese P. Giacometti G.M. Moya I. J. Photochem. Photobiol. 1991; 6: 381-394Google Scholar, 28Peterman E.J.G. Dukker F.M. van Grondelle R. Van Amerongen H. Biophys. J. 1995; 69: 2670-2678Abstract Full Text PDF PubMed Scopus (158) Google Scholar). Here we report on monomeric proteins. Mutations at sites A1 and A4 affect Glu/Arg ionic pairs in the helix A/helix B cross. The spectrum of the A4 mutant shows a red shift of the major (Chl a) peak by 1.2 nm with respect to the WT and higher amplitude of the 652 nm shoulder (Fig. 3 A). The second derivative analysis showed a decreased amplitude of a spectral form around 675 nm (Fig. 3 A.1). Mutation at site A1 (E180L/R70I) yielded a large blue shift (4 nm) of the Chl a peak, and the ratio of the amplitudes of the Chl a versus Chl b peaks was lower with respect to the WT. Second derivative analysis clearly shows perturbation of the red-most signal at around 680 nm (Fig. 3,A and A.1). Mutations at sites A2 and A5 also affect two Chls close to the center of the LHCII structure (Fig. 1). The A2 mutant spectrum (Fig.3 B) shows a blue shift of 2 nm with respect to the WT because of the complete loss of the red most signal at around 680 nm (Fig. 3 B.1). The mutation in the A5 site (H68I) yields a spectrum rather similar to the A4 mutation showing a decrease in the ratio between the Chl a and Chl b peaks with respect to the WT, because of the loss of a spectral form absorbing at 674–675 nm (Fig. 3,B and B.1). The Chl bound to site A6 has been proposed to be coordinated through the Gly78 peptidyl-carbonyl. The absorption spectrum of the P82V mutant is identical to that of WT, suggesting that if this is the ligand of Chl in site A6, the availability of this group is not essential to Chl binding (Fig. 3, C and C.1). Mutations B5.1, B5.2, B6.1, and B6.2 are targeted to ligands on helix C. Their absorption spectra show similar characteristics, suggesting that the chromophores affected by these mutation might be part of a common pool (Fig. 3, D and D.1). The main feature is a decrease of the Chl b peak at around 652 nm with the amplitude of the effect being in the order B5.1 (E139L) < B6.1 (Q131L) < B5.2 (E139L/R142L). Differences with respect to the WT spectrum are also evident in the Chl a region, where blue shifts of the peak, by 2, 2, and 3 nm, respectively, were observed. The second derivative analysis also shows that upon removal of bulk 652 nm absorption, a 646 nm signal is revealed in the B5.2 (E139L/R142L) mutant, suggesting that this absorption form is associated to a site located in a protein domain not affected by helix C mutations (Fig. 3 D.1). Differences are also detected in the Chl a spectral region as changes in the relative amplitude of the different absorption forms (Fig.3 D.2). The B6.2 (Q131E) mutation is intended to substitute a Chl-binding residue with another putative one present, in the same position, in homologous proteins like CP29, CP26, or LHCI. This mutation leads to a decreased Chl b absorption (652 nm) accompanied by increased absorption at around 675 nm (Fig. 3 D), suggesting that the B6 site decreases its affinity for Chl b in favor of Chl a. The spectrum is otherwise similar to WT. The targets of B3 and A3 mutations are located in the D helix domain. The absorption spectra of both mutant proteins shows a decreased Chl b (652 nm) absorption (Fig. 3 E). Nevertheless, the trough between Chl a and Chl b peaks became more evident, suggesting that blue Chl a absorption forms at around 663–665 nm are depleted as indicated by the second derivative analysis (Fig. 3 E.1). The spectral effects displayed by mutant proteins clearly indicate that mutations are specific for different absorption forms covering the whole of the LHCII spectrum. This suggests that these constructions can be used for correlating the absorption forms with specific chromophores in LHCII. Previous work on carotenoid-binding sites of LHCII showed that maize Lhcb1 has three sites tightly binding xanthophylls in agreement with the results with LHCII from thylakoid membranes (8Bassi R. Pineau B. Dainese P. Marquardt J. Eur. J. Biochem. 1993; 212: 297-303Crossref PubMed Scopus (350) Google Scholar, 9Connelly J.P. Muller M. Bassi R. Croce R. Holzwarth A.R. Biochemistry. 1997; 36: 281-287Crossref PubMed Scopus (116) Google Scholar, 10Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Two of them, located in the helix A/helix B cross as detected by electron crystallography (5Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1542) Google Scholar), can bind either lutein or violaxanthin in a 1.8 to 0.2 ratio. In this work, because of the slightly different conditions of reconstitution we obtained 1.68 ± 0.02 lutein and 0.32 ± 0.03 violaxanthin for WT LHCII. The third site, not resolved in the structure, is selective for neoxanthin. Pigment composition of WT and mutant proteins was determined by HPLC analysis and extrapolation from the absorption spectra of the acetone extracts. This combined approach proved to be effective in minimizing errors in the Chl/car ratio (9Connelly J.P. Muller M. Bassi R. Croce R. Holzwarth A.R. Biochemistry. 1997; 36: 281-287Crossref PubMed Scopus (116) Google Scholar). Because the lutein-binding sites are located in the loops between α-helices (29Pichersky E. Jansson S. Ort D.R. Yokum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer, Dordrecht, The Netherlands1996: 507-521Google Scholar), which are not targeted by mutations, we tentatively used lutein (1.68 mol/mole of protein) as a reference. The results are shown in TableII. The validity of the assumption of 1.68 luteins/polypeptide was verified by direct measurement of the Chl to protein stoichiometry as previously performed in the homologous protein CP29 (26Bassi R. Croce R. Cugini D. Sandonà D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10056-10061Crossref PubMed Scopus (191) Google Scholar). However, the amount of material and the number of repetitions needed for this determination allowed reliable results only on mutant proteins obtained with high yield (TableIII). These results support the validity of the data in Table II, although a discrepancy was found in the case of the A4 mutant.Table IIPigment composition of WT and mutant LHCII proteins as determined by HPLC analysis and acetone extract fittingChl a/bChl totalChl aChl bLuteinneoviolaΔChl aΔChl bΔChlWT1.412751.681.050.32A1 (E180L/R70I)1.0810.55.455.051.651.080.07−1.5−1.5A2 (N183L)1.449.45.543.841.680.840.07−1.5−1.2−2.7A3 (Q197L)1.45116.514.491.7210.2−0.5−0.5−1.0A4 (E65L/R185L)1.5111.77.074.671.681.170.19−0.3−0.3*A4 (E65L/R185L)1.51106.023.981.4310.16−1.0−1.0−2.0A5 (H68I)1.1911651.671.10.12−1−1.0A7 (P82V)1.412751.680.850.2B3 (H212V)1.43116.474.531.680.830.16−0.5−0.5−1.0B5.1 (E139L)1.57106.13.91.680.640.1−0.9−1.1−2.0B5.2 (E139L/R142L)3.7186.31.71.660.040.09−0.7−3.3−4.0B6.1 (Q131L)1.82106.453.541.670.480.06−0.5−1.5−2.0B6.2 (Q131E)1.18126.55.51.681.00.15−0.5+0.5Values are in mol/mol LHCII polypeptide as calculated in the assumption of 1.68 lutein/polypeptide chain. The data are the averages of three measurements. Open table in a new tab Table IIIStoichiometry of chlorophyll to protein as determined by the ninhydrin methodProteinChl/polypeptideWT12.0 ± 0.4A2 (N183L)9.5 ± 0.6A3 (Q197L)11.1 ± 0.3A4 (E65L/R185L)9.8 ± 0.3A5 (H68I)11.0 ± 0.3A7 (P82V)11.8 ± 0.5B3 (H212V)11.2 ± 0.4B6.2 (Q131E)11.8 ± 0.2The measurements are the averages of 12 measurements on three preparations. Values are expressed in moles (see “Experimental Procedures”). Open table in a new tab Values are in mol/mol LHCII polypeptide as calculated in the assumption of 1.68 lutein/polypeptide chain. The data are the averages of three measurements. The measurements are the averages of 12 measurements on three preparations. Values are expressed in moles (see “Experimental Procedures”). All the mutant proteins showed a lower Chl content per 1.68 luteins than the WT protein with the exception of the P82V (site A7), which had the same composition as the WT, and the Q131E (site B6), which showed a higher Chl a content, suggesting the mutation increased the affinity for Chl a in site B6. The A4 mutant showed very little change in pigment composition on a lutein basis. However, strong effects on the absorption spectrum (Fig. 3 B) and the Chl/protein stoichiometry of 10 (Table III) consistently indicated the loss of two Chl. We therefore recalculated the pigment composition of the A4 mutant on the basis of 10 Chl (a+b); these values are given as (*A4) in TableII. From this new normalization it appears that this mutant lost part of its lutein, whereas the neoxanthin content was not affected. Three mutant proteins were found to bind 11 chlorophylls; the A5 mutant lost Chl a only, whereas the A3 and B3 mutants appear to loose both Chl a and Chl b, thus leading to the tentative conclusion that site A5 binds Chl a, while B3 and A3 sites can be occupied by either Chl a or Chl b with roughly equal probability. Somewhat similar is the case of mutant A1, which appears to lose Chl a only in the amount of 1.5 mol/mole of protein. This result suggests not only that site A1 is occupied by Chl a but also that a neighbor site occupied by Chl a is affected. This is probably site B1, which is the closest one for which a specific binding residue was not detected. These results are in agreement with data obtained in the homologous protein CP29 (26Bassi R. Croce R. Cugini D. Sandonà D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10056-10061Crossref PubMed Scopus (191) Google Scholar). The most striking result, however, was the loss of more than one and up to four Chl molecules in the case of five mutant proteins (B5.1, B5.2, B6.1, A2, and A4). This is evident not only from the stoichiometry on lutein or protein basis but also from the Chl a/b ratio. The expected values in the case of a single Chl a or Chl b loss are respectively of 1.2 and 1.75, whereas values up to 3.7 from the WT result of 1.4 were obtained. This indicates that" @default.
• W2006823377 created "2016-06-24" @default.
• W2006823377 creator A5020140482 @default.
• W2006823377 creator A5021235936 @default.
• W2006823377 creator A5030196756 @default.
• W2006823377 creator A5036165713 @default.
• W2006823377 creator A5037761633 @default.
• W2006823377 date "1999-11-01" @default.
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• W2006823377 title "Chlorophyll Binding to Monomeric Light-harvesting Complex" @default.
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