In VitroReconstitution of the Recombinant Photosystem II Light-harvesting Complex CP24 and Its Spectroscopic Characterization
1998; Elsevier BV; Volume: 273; Issue: 27 Linguagem: Inglês
10.1074/jbc.273.27.17154
ISSN1083-351X
AutoresAldo Pagano, Gianfelice Cinque, Roberto Bassi,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe light-harvesting chlorophyll a/bprotein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophylla, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or β-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyllb and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophylla to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/bratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions.The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophylla molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll babsorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a. The light-harvesting chlorophyll a/bprotein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophylla, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or β-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyllb and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophylla to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/bratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions. The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophylla molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll babsorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a. In higher plants, chloroplasts, chlorophyll, and carotenoid molecules are noncovalently bound to specific transmembrane proteins to form light-harvesting complexes called LHCI 1The abbreviations used are: LHCI, light-harvesting complex of PSI; LHCII, light-harvesting complex of PSII; Chl, chlorophyll; CP, chlorophyll protein; DM, dodecylmaltoside; FWHM, full-width half-maximum; LiDS, lithium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PSI, photosystem I; PSII, photosystem II; rCP24 and rCP29, recombinant CP24 and CP29, respectively, reconstituted from the apoprotein derivatives overproduced in bacteria; nCP24, native CP24; HPLC, high pressure liquid chromatography; FPLC, fast protein liquid chromatography. and LHCII. These antenna complexes efficiently capture the light and deliver the excitation energy, respectively, to photosystem I (PSI) and II (PSII) reaction centers, where electron transport occurs, yielding atrans-thylakoid pH gradient, ATP synthesis, and NADP+ reduction. The photosystem II light-harvesting complex has been extensively investigated and shown to be composed of four chlorophyll a/b proteins, the major complex (LHCII) binding about 65% of PSII chlorophyll and three minor complexes (called CP24, CP26, and CP29) that together bind about 15% of total PSII chlorophyll (1Dainese P. Bassi R. J. Biol. Chem. 1991; 266: 8136-8142Abstract Full Text PDF PubMed Google Scholar). These minor chlorophyll proteins appear to be involved in the dissipation of the chlorophyll excitation energy needed to prevent overexcitation and photoinhibition of PS II (see Ref. 2Bassi R. Sandonà D. Croce R. Physiol. Plant. 1997; 100: 769-779Crossref Google Scholar for a review). It was shown that more than 80% of the xanthophyll violaxanthin is associated to minor complexes in maize (3Bassi R. Pineau B. Dainese P. Marquardt J. Eur. J. Biochem. 1993; 212: 297-303Crossref PubMed Scopus (352) Google Scholar) and that CP24 is the one with the highest violaxanthin binding capacity. This pigment is involved in the major photoprotection mechanism in plants, known as "nonphotochemical quenching" (4Demmig-Adams B. Biochim. Biophys. Acta. 1990; 1020: 1-24Crossref Scopus (1415) Google Scholar), through the operation of a xanthophyll cycle by which it is deepoxidated to antheraxanthin and zeaxanthin (5Yamamoto H. Bassi R. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 539-563Google Scholar). The intermediate location of minor complexes between the reaction center and the major LHCII complex (6Bassi R. Dainese P. Eur. J. Biochem. 1992; 204: 317-326Crossref PubMed Scopus (165) Google Scholar) makes them well suited for regulating the excitation energy supply to PSII or its dissipation. The structural bases for the regulatory properties of the minor chlorophyll proteins are mostly unknown due to the difficulties in the isolation of these proteins in sufficient amounts and in their native form. To overcome this problem, we have reconstituted the CP24 holoprotein using overexpressed apoprotein from Escherichia coli and purified pigments. Its characterization allowed us to obtain previously unavailable information on the number of chromophores bound to this protein and opens the way to the mutational analysis of this PSII subunit in both its protein moiety and the chromophores bound. As an example of the usefulness of recombinant pigment-proteins, we have used the recombinant CP24 in order to address the problem of Chl a and Chl b absorption in antenna proteins; while only two chemically distinct chlorophyll species are present, many optical transitions (spectral forms) are commonly observed in the Qy absorption region (7Hemelrijk P.W. Kwa S.L.S. van Grondelle R. Dekker J.P. Biochim. Biophys. Acta. 1992; 1098: 159-166Crossref Scopus (152) Google Scholar, 8Kwa S.L.S. Groeneveld F.G. Dekker J.P. van Grondelle R. van Amerongen Lin H. Struwe W.S. Biochim. Biophys. Acta. 1992; 1101: 143-146Crossref Scopus (36) Google Scholar, 9Reddy N.R.S. van Amerongen H. Kwa S.L.S. van Grondelle R. Small G.J. J. Phys. Chem. 1994; 98: 4729-4735Crossref Scopus (60) Google Scholar, 10Jennings R.C. Bassi R. Garlaschi F.M. Dainese P. Zucchelli G. Biochemistry. 1993; 32: 3203-3210Crossref PubMed Scopus (92) Google Scholar, 11Zucchelli G. Dainese P. Jennings R.C. Breton J. Garlaschi F.M. Bassi R. Biochemistry. 1994; 33: 8982-8990Crossref PubMed Scopus (59) Google Scholar, 12Giuffra E. Zucchelli G. Sandonà D. Croce R. Cugini D. Garlaschi F.M. Bassi R. Jennings R. Biochemistry. 1997; 36: 12984-12993Crossref PubMed Scopus (63) Google Scholar). Lack of progress in understanding this spectroscopic heterogeneity has been mainly due to the absence of experimental techniques to enable selective modification of the optical transitions. Also, it has not been possible to assign particular transitions to Chl a or Chl b though it is generally assumed that the shorter wavelength bands are associated with Chl b. Analysis of a recombinant CP24 complex binding mainly Chl b allowed us to identify four Gaussian subbands peaking at 638, 645, 652, and 659 nm, which are also present in the native complex, thus identifying the principle components of Chl babsorption in the antenna protein environment. To overexpress plant CP24 in E. coli, the maizeLhcb6 cDNA (13Dainese P. Bergantino E. Sechi S. Bassi R. Pichersky E. Murata N. Developments in Photosynthesis Research. 1. Kluwer Academic Publishers, Dordrecht, The Netherlands1992: 199-202Google Scholar) was subcloned into an expression vector of the pDS series (14Bujard H. Gentz R. Lanzer M. Stuber D. Muller M. Ibrahimi I. Hauptle M.T. Dobberstein B. Methods Enzymol. 1987; 155: 416-433Crossref PubMed Scopus (230) Google Scholar). A clone was obtained by polymerase chain reaction mutagenesis of Lhcb6 DNA. The construct pDS 12–24αε (Fig. 1 A) was obtained by using two primers (5′-CCGCGCGCAGATCTTCGCC-3′ (carrying the BglII site) and 5′-TCTGATCCCATGCATCCGTACGTC-3′ (carrying the NsiI site)), allowing the amplification of a 733-base pair fragment spanning the full coding region. After digestion with BglII andNsiI, the resulting fragment was subcloned into the pDS-RBS II expression vector. Thus, the pDS 12–24αε construct codes for a protein containing four additional residues (MRIA …) extending the N teminus as compared with the native protein (Fig. 1 B). Plasmids were constructed using a standard molecular cloning procedures (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Bacterial hosts were E. coli (SG13009 strain) (16Gottesman S. Halpern E. Trisler P. J. Bacteriol. 1981; 148: 265-273Crossref PubMed Google Scholar). CP24 apoprotein was isolated from the SG13009 strain transformed with the construct according to the protocols in Refs. 17Nagai K. Thogersen H.C. Methods Enzymol. 1987; 153: 461-481Crossref PubMed Scopus (367) Google Scholarand 18Paulsen H. Rümler U. Rüdiger W. Planta. 1990; 181: 204-211Crossref PubMed Scopus (228) Google Scholar. Total pigment extracts were obtained by extracting thylakoids of wild-type barley with 80% acetone. Extracts of Chl a and carotenoids were obtained by using thylakoids from the Chl b-less mutant chlorina f2(19Simpson D.J. Carlsberg Res. Commun. 1979; 44: 305-336Crossref Scopus (103) Google Scholar). Chl b and carotenoids were obtained by preparative HPLC using a reverse phase column (PHENOMENEX, Torrance, CA) bondclone 10 C18 (7.8 × 300 mm) using 82% acetone as eluent. Reconstituted complexes were analyzed for their pigment composition after 80% acetone extraction as described previously (3Bassi R. Pineau B. Dainese P. Marquardt J. Eur. J. Biochem. 1993; 212: 297-303Crossref PubMed Scopus (352) Google Scholar). During all of the procedures, care was taken to protect pigments from light and oxygen. The concentration of pigments was determined spectroscopically according to Ref. 20Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4891) Google Scholar for chlorophylls and using the extinction coefficients given by Davies (21Davies B.H. Goodwin T.W. Chemistry and Biochemistry of Plant Pigments. 2. Academic Press, London1976: 38-165Google Scholar) for xanthophylls. The concentration of carotenoid mixtures was estimated on the basis of an average molar extinction coefficient of 1.4 × 10−5 at 444 nm. Pigment composition of chlorophyll proteins was determined by HPLC analysis according to Ref. 22Gilmore A.M. Yamamoto H.Y. J. Chromatogr. 1991; 543: 137-145Crossref Scopus (415) Google Scholar. Chl a/b ratio and Chl/carotenoid ratio was also determined by fitting the spectrum of ethanol extracts with the spectra of purified pigments. The reconstitution procedure largely followed the one designed for LHCII (18Paulsen H. Rümler U. Rüdiger W. Planta. 1990; 181: 204-211Crossref PubMed Scopus (228) Google Scholar, 23Plumley F.G. Schmidt G.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 146-150Crossref PubMed Google Scholar). In the basic procedure, 400 μg of protein of CP24 apoprotein isolated from bacteria was solubilized in 1000 μl of a buffer containing 100 mm Tris-HCl (pH 10), the protease inhibitors 6-aminocaproic acid (5 mm) and benzamidine (1 mm), 12.5% sucrose, 2% LiDS by heating to 100 °C for 3 min and sonication. After the addition of 100 mm dithiothreitol and the pigment solution in 70 μl of ethanol, the mixture was sonicated again. Reconstitution was achieved by three subsequent cycles of freezing (1 h, −20 °C) and thawing (30 min, room temperature). Octyl β-D-glucopyranoside was then substituted for LiDS by precipitation of the potassium dodecyl sulfate following the addition of 4% KCl, incubation for 15 min in ice, and centrifugation (10 min at 13,000 × g). The mixture was then loaded on a 12-ml sucrose gradient (0.1–1 m) containing 10 mm Hepes, pH 7.6, and 0.06% DM and centrifuged for 17 h at 254,000 ×g in a Beckman SW 41 rotor. The lower green band (at about 0.4 m sucrose) contained the reconstituted complex and was harvested with a syringe. Maximal yield was obtained with chlorophyll:protein molar ratios between 40 and 80, corresponding to a 5–10-fold excess of pigments. In this work, the carotenoid concentration was maintained at 60 μg/ml. The green bands from the sucrose gradient were then subjected to chromatography on a Fractogel EMD-DMAE column (15 × 150 mm) (Merck). After loading, the column was washed with 0.025% DM (70 min at 1 ml/min). The chlorophyll-protein was then eluted by applying a 0–500 mm NaCl gradient. The peak fractions were concentrated by Centricon centrifugation and loaded into a glycerol gradient (10–25%) containing 0.06% DM and 10 mm Hepes, pH 7.6. The gradient was spun overnight in SW 60 Beckman rotor at 450,000 × g, yielding a faint upper band of free pigments and a lower band with the chlorophyll-protein, which was frozen in liquid nitrogen and kept at −80 °C until use. CP24 was isolated from maize PSII membranes as described previously (1Dainese P. Bassi R. J. Biol. Chem. 1991; 266: 8136-8142Abstract Full Text PDF PubMed Google Scholar, 24Dainese P. Hoyer-Hansen G. Bassi R. Photochem. Photobiol. 1990; 51: 693-703Crossref PubMed Google Scholar). Mildly denaturing electrophoresis was according to Ref. 25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212372) Google Scholar but at 4 °C and with 20% glycerol in resolving and stacking gel. Denaturing electrophoresis was according to Ref. 26Shägger H. von Jagow H. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10686) Google Scholar. Protein concentration was determined by the bicinchoninic acid method (27Smith 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 (19134) Google Scholar). Densitometry was performed with a Bio-Rad 600 scanning densitometer after staining of the gel and destaining according to Ref. 28Ball E.H. Anal. Biochem. 1986; 155: 23-27Crossref PubMed Scopus (109) Google Scholar. Absorption spectra were obtained using an SLM-Aminco DW-2000 spectrophotometer at room temperature. Fluorescence excitation and emission spectra were obtained by using a Jasco-600 spectrofluorimeter. Samples were in 10 mm Hepes, pH 7.6, 0.06% DM, 20% glycerol. Chlorophyll concentration was about 10 μg/ml for absorption measurements and 0.01 μg/ml for fluorescence measurements. Emission and excitation spectra were corrected for instrumental response. Analysis of fluorescence spectra was performed according to Stepanov as previously reported (12Giuffra E. Zucchelli G. Sandonà D. Croce R. Cugini D. Garlaschi F.M. Bassi R. Jennings R. Biochemistry. 1997; 36: 12984-12993Crossref PubMed Scopus (63) Google Scholar, 29Stepanov B.I. Sov. Phys. Dokl. 1957; 2: 81-84Google Scholar). Circular dichroism spectra were recorded with a Jasco J-600 spectropolarimeter as previously reported (12Giuffra E. Zucchelli G. Sandonà D. Croce R. Cugini D. Garlaschi F.M. Bassi R. Jennings R. Biochemistry. 1997; 36: 12984-12993Crossref PubMed Scopus (63) Google Scholar). Similarly to a described method (11Zucchelli G. Dainese P. Jennings R.C. Breton J. Garlaschi F.M. Bassi R. Biochemistry. 1994; 33: 8982-8990Crossref PubMed Scopus (59) Google Scholar), the decomposition of the absorbanceversus wavelength was obtained by a nonlinear least squares fitting code (OriginTM; MicroCal. Software Inc., Northampton, MA). Here, a linear combination of maximum 10 symmetric Gaussians (eight absorption bands plus two border ones for tails' adjustment) was considered in the χ2 minimization (error considered by counting statistics) by means of the Levenberg–Marquardt algorithm (30Press W.H. Flannery B.P. Teukolsky S.A. Vetterling W.T. Numerical Recipes. Cambridge University Press, Cambridge1986Google Scholar). All of the parameters were kept free and always unconstrained in the analysis; the fitting procedure was reproducible when starting from a reasonable initial choice for subband FWHM (less than 10 nm shared by all subbands) and peaks (within the 630–720 wavelength range). Less than 50 iterations were necessary for achieving the subband positioning, and, after assigning the FWHM to the different Gaussians, the convergence was estimated by a χ2variation down to a few percentages in successive single iterations. The E. colihost cells SG13009 (16Gottesman S. Halpern E. Trisler P. J. Bacteriol. 1981; 148: 265-273Crossref PubMed Google Scholar) are a K12-derived strain. They were transformed with the pREP4 plasmid, which carries the kanamycin selection and thelacI gene, encoding the Lac repressor, thus allowing a tight control over the level of expression (Qiagen). The construct is described in Fig. 1 A, and the sequence coded is shown in Fig. 1 B. The bacterial strain transformed with the pDS 12–24αε produced, upon induction with 2 mm isopropyl-1-thio-β-d-galactopyranoside, the protein of the expected molecular weight as detected by Western blotting with a CP24 antibody (31Di Paolo M.L. Dal Belin-Peruffo A. Bassi R. Planta. 1990; 181: 275-286Crossref PubMed Scopus (52) Google Scholar) (Fig. 1, C andD). The best results were obtained after 6–7 h of isopropyl-1-thio-β-d-galactopyranoside induction in superbroth. In all conditions tested, the expressed protein reached 10% of the total protein extract. Fractionation of the bacterial cells by the method in Ref. 17Nagai K. Thogersen H.C. Methods Enzymol. 1987; 153: 461-481Crossref PubMed Scopus (367) Google Scholar showed that the expression products are accumulated in inclusion bodies, as shown for LHCII (18Paulsen H. Rümler U. Rüdiger W. Planta. 1990; 181: 204-211Crossref PubMed Scopus (228) Google Scholar, 32Cammarata K.V. Schmidt G.W. Biochemistry. 1992; 31: 2779-2789Crossref PubMed Scopus (53) Google Scholar) and CP29 (33Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar). Repeated washings of this pelletable fraction yielded 80% pure CP24 apoprotein as judged by polyacrylamide electrophoresis. The protein could be easily purified to homogeneity by preparative isoelectric focusing, but most of the experiments here described were performed with the 80% pure preparation without affecting the efficiency of the reconstitution. All experiments described in this study have been performed with rCP24 purified as shown in Fig. 1 C. Triton X-100-washed inclusion bodies (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) were extracted with 80% acetone in order to remove residual Triton and solubilized in 2% LiDS in a bath sonicator followed by boiling. Pigments were then added from stock solutions in ethanol, and the reconstitution procedure was carried out as described under "Experimental Procedures." In this experiment, a Chl a to Chl b ratio of 1.6, similar to the ratio in the native complex (1Dainese P. Bassi R. J. Biol. Chem. 1991; 266: 8136-8142Abstract Full Text PDF PubMed Google Scholar), was used, and the mixture contained a total carotenoid extract from thylakoids, thus including β-carotene, lutein, neoxanthin, violaxanthin. Following reconstitution, the mixture was ultracentrifuged through a 0.1–1 m sucrose gradient, yielding a green band at 0.4 m sucrose, well separated from the free pigment band on the upper part of the tube at 0.1–0.2m sucrose (Fig. 2 A). The lower green band was harvested with a syringe and subjected to DMAE chromatography. Once bound to the column in 10 mm Hepes-KOH, pH 7.6, 50 mm NaCl, 0.03% DM, the nonspecifically bound pigments were washed from the complexes and the column with 10 mmHepes-KOH, pH 7.6, 50 mm NaCl, 0.03% DM at 1 ml/min. This procedure removed about 50% of the chlorophyll loaded in the column but not the protein. The protein was eluted at 200 mm NaCl. When centrifuged on a 10–25% glycerol gradient, the eluted protein appeared as a single green band without residual free pigments (Fig. 2 B). When isolated from thylakoid membranes, CP24 binds lutein and violaxanthin but not neoxanthin (3Bassi R. Pineau B. Dainese P. Marquardt J. Eur. J. Biochem. 1993; 212: 297-303Crossref PubMed Scopus (352) Google Scholar). In order to elucidate the role of different xanthophyll species in refolding of CP24, we have carried on the reconstitution procedure with pigment mixtures differing in xanthophyll composition. In each experiment, one xanthophyll species was omitted from the mixture. Alternatively, reconstitution without xanthophylls (only Chl a and Chl b) or with the complete carotenoid set was performed. The stability of the resulting complex was assayed at two levels of stringency: sucrose gradient ultracentrifugation (Fig. 3 A) and mildly denaturing LiDS-PAGE (Fig. 3 B). When xanthophylls were omitted from the mixture, a green band was still obtained after sucrose gradient ultracentrifugation, having the same mobility as the control sample with a complete xanthophyll supply, suggesting a partial folding was obtained even in the absence of carotenoids. However, this complex did not survive LiDS-PAGE. The samples refolded in the presence of two xanthophylls yielded a green band stable both in sucrose gradients and in LiDS-PAGE, and the yields were comparable with each other and with the control sample prepared in the presence of the three xanthophylls. Particularly interesting is the case of sample A, where a stable complex was obtained in the absence of lutein. This result is at variance with previous results with LHCII and CP29 (18Paulsen H. Rümler U. Rüdiger W. Planta. 1990; 181: 204-211Crossref PubMed Scopus (228) Google Scholar, 33Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar) in which lutein was shown to be essential for stability. The data reported above show that CP24 can be successfully refolded in vitro. The use of this recombinant protein for mutation analysis is, however, more informative if its characteristics closely reflect those of the native complex extracted from thylakoid membranes. We optimized the conditions for the recombinant protein to reproduce the chlorophyll a/b ratio of native CP24. For comparison, native CP24 was purified from maize PSII membranes as described previously (1Dainese P. Bassi R. J. Biol. Chem. 1991; 266: 8136-8142Abstract Full Text PDF PubMed Google Scholar), and it showed an a/b ratio of 1.2. A series of reconstitution mixtures were performed with decreasing Chla/b ratios, namely 8.0, 5.8, 2.0, 1.5, 1.0 and 0.001. The last preparation was intended to contain only Chl b. However, HPLC analysis of the reconstitution mixture showed 1:1000 contamination by Chl a. This experiment was performed on a semipreparative scale, following the procedure in Fig. 2, finally obtaining, from glycerol gradient ultracentrifugation, sufficient amounts of the complex for spectroscopic and biochemical analysis. In Fig. 4, a plot is shown of the dependence of the Chl a/b ratio in the complex on the Chla/b ratio in the reconstitution mixture; although the reconstitution with Chl b only yielded a stable complex having a Chl a/b ratio of 0.12, the Chl a/b ratio of the reconstituted complex rises steeply to about 1 with increasing Chl a availability and reaches a plateau between ratios of 2.0 and 5.8. Within this range, a ratio of 1.0 for bound Chla and Chl b was obtained. When a large Chla excess was applied (Chl a/b ratio of 8) it was possible to obtain a Chl a/b ratio in the reconstituted complex of 1.4. As a comparison, the data previously obtained for CP29 (12Giuffra E. Zucchelli G. Sandonà D. Croce R. Cugini D. Garlaschi F.M. Bassi R. Jennings R. Biochemistry. 1997; 36: 12984-12993Crossref PubMed Scopus (63) Google Scholar, 33Giuffra E. Cugini D. Croce R. Bassi R. Eur. J. Biochem. 1996; 238: 112-120Crossref PubMed Scopus (116) Google Scholar) are also reported, showing that the two proteins clearly differed in their affinity for Chl a and b. From this comparison, the two complexes with Chl a/b of 1.0 and 1.4 most closely resemble native CP24. In order to further characterize the recombinant CP24 complex (a/b = 1.4 and a/b = 1.0), we determined the chlorophyll to protein stoichiometry. Toward this end, we used a highly purified LHCII preparation (24Dainese P. Hoyer-Hansen G. Bassi R. Photochem. Photobiol. 1990; 51: 693-703Crossref PubMed Google Scholar) as a standard protein binding 12.6 ± 0.1 chlorophylls/polypeptide (1Dainese P. Bassi R. J. Biol. Chem. 1991; 266: 8136-8142Abstract Full Text PDF PubMed Google Scholar, 34Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1552) Google Scholar). The chlorophyll concentration of the LHCII and of CP24 samples was carefully determined by HPLC analysis, and aliquots of the samples corresponding to different amounts of chlorophyll were loaded on an SDS-PAGE gel. After running, the gel was stained for quantitative analysis according to Ball (28Ball E.H. Anal. Biochem. 1986; 155: 23-27Crossref PubMed Scopus (109) Google Scholar), and the Coomassie Blue binding to CP24 and LHCII gel bands was determined by densitometry and by elution of stain and spectrophotometric determination. The Coomassie Blue-stained gel and the resulting plot is shown in Fig. 5, B and C. Pigment binding to rCP24 was 0.79 with respect to LHCII. Once corrected for the sequence-specific difference in Coomassie Blue binding between CP24 and LHCII (1Dainese P. Bassi R. J. Biol. Chem. 1991; 266: 8136-8142Abstract Full Text PDF PubMed Google Scholar), a value of 10 chlorophyll (a plus b) mol per mol of CP24 apoprotein was obtained. The results of HPLC analysis of pigments extracted from native and recombinant CP29 proteins are reported in TableI. Both the native and the recombinant protein contained, besides Chl a and b, the xanthophylls lutein and violaxanthin but not neoxanthin and β-carotene, although the latter carotenoids were present in the reconstitution mixture in the same amounts as lutein and violaxanthin. Per 10 chlorophylls (a plus b), two xanthophylls were found per rCP24 polypeptide. This is consistent with the value of two xanthophyll molecules per polypeptide found in the homologous protein CP29 (35Pesaresi P. Sandonà D. Giuffra E. Bassi R. FEBS Lett. 1997; 402: 151-156Crossref PubMed Scopus (71) Google Scholar). Table I also reports HPLC analysis of the rCP24 proteins with Chl a/b ratio of 0.12, obtained by refolding with Chl a/b = 0.001. It is shown that the specificity of carotenoid binding is retained irrespective of the binding of Chla or Chl b, since neoxanthin was never present in the proteins. If a value of two xanthophyll molecules per polypeptide is assumed (as in the rCP24 1.4 and rCP24 1.0), then rCP24 0.12 binds one Chl a and eight Chl b as shown by the Chl:xanthophyll ratio, which is 4.5, thus speaking for nine bound Chl rather than 10. These data are summarized in TableII.Table IHPLC analyses of nCP24, rCP24 1.0, rCP24 1.4, rCP24 0.12, and LHCIISampleChl a/b ratio (during folding)Chl a/b ratio (in the complex)ViolaxanthinLuteinNeoxanthinChlorophyllbChlorophyll anCP241.2217.519.80.0683.4100rCP24 1.02.00.9611.329.70.05103.8100rCP24 1.05.80.9512.527.60.06104.7100rCP24 1.48.01.4211.822.10.0970.7100rCP24 0.120.0010.1279.6120.40.09800100Native LHCII1.451.625.711.971.51
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