Assembly of the Major Light-harvesting Chlorophyll-a/b Complex
2006; Elsevier BV; Volume: 281; Issue: 35 Linguagem: Inglês
10.1074/jbc.m604828200
ISSN1083-351X
AutoresStephan Hobe, I. Trostmann, Stefan Raunser, Harald Paulsen,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoThe major light-harvesting chlorophyll-a/b complex in most higher plants contains three carotenoids, lutein, neoxanthin, and violaxanthin. How these pigments are assembled into the complex during its biogenesis is largely unknown. Here we show that neoxanthin but not lutein can dissociate from the fully assembled complex. Its equilibrium binding constant in a detergent system (0.1% n-dodecyl-β-d-maltoside) was determined to be ≥ 106 m–1. Neoxanthin insertion into light-harvesting chlorophyll-a/b complex prefolded from overexpressed apoprotein (Lhcb1*2 from Pisum sativum) in the presence of chlorophylls a, b, and lutein as the sole carotenoid is kinetically controlled by an activation energy barrier of ∼120 kJ mol–1. This is the first thermodynamic and kinetic description of a binding equilibrium between a non-covalently bound pigment of the photosynthetic apparatus and its protein complex. Dissociation of neoxanthin from the major light-harvesting chlorophyll-a/b complex upon temperature increase is discussed in terms of providing a readily available substrate pool for synthesizing abscisic acid as part of a heat and drought stress response. The major light-harvesting chlorophyll-a/b complex in most higher plants contains three carotenoids, lutein, neoxanthin, and violaxanthin. How these pigments are assembled into the complex during its biogenesis is largely unknown. Here we show that neoxanthin but not lutein can dissociate from the fully assembled complex. Its equilibrium binding constant in a detergent system (0.1% n-dodecyl-β-d-maltoside) was determined to be ≥ 106 m–1. Neoxanthin insertion into light-harvesting chlorophyll-a/b complex prefolded from overexpressed apoprotein (Lhcb1*2 from Pisum sativum) in the presence of chlorophylls a, b, and lutein as the sole carotenoid is kinetically controlled by an activation energy barrier of ∼120 kJ mol–1. This is the first thermodynamic and kinetic description of a binding equilibrium between a non-covalently bound pigment of the photosynthetic apparatus and its protein complex. Dissociation of neoxanthin from the major light-harvesting chlorophyll-a/b complex upon temperature increase is discussed in terms of providing a readily available substrate pool for synthesizing abscisic acid as part of a heat and drought stress response. The photosynthetic apparatus in the thylakoid membrane of green plants contains light-harvesting complexes enhancing its capacity to absorb light quanta that are then converted into chemical potential by the reaction centers. The major light-harvesting complex of photosystem II, LHCIIb, 2The abbreviations used are: LHCIIb, major light harvesting chlorophyll a/b complex of photosystem II; ABA, abscisic acid; β-DM, n-dodecyl-β-d-maltoside; LL·LHCIIb, partially pigmented LHCIIb carrying two molecules of Lu; Lu, lutein; NLL·LHCIIb, fully pigmented LHCIIb carrying two molecules of Lu as well as one Nx; Nx, neoxanthin; Vx, violaxanthin; Zx, zeaxanthin; HPLC, high-performance liquid chromatography.2The abbreviations used are: LHCIIb, major light harvesting chlorophyll a/b complex of photosystem II; ABA, abscisic acid; β-DM, n-dodecyl-β-d-maltoside; LL·LHCIIb, partially pigmented LHCIIb carrying two molecules of Lu; Lu, lutein; NLL·LHCIIb, fully pigmented LHCIIb carrying two molecules of Lu as well as one Nx; Nx, neoxanthin; Vx, violaxanthin; Zx, zeaxanthin; HPLC, high-performance liquid chromatography. is assembled in its trimeric form, equipped with 14 chlorophylls and 4 xanthophylls per apoprotein (1Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1350) Google Scholar, 2Standfuss J. Van Scheltinga A.C.T. Lamborghini M. Kühlbrandt W. EMBO J. 2005; 24: 919-928Crossref PubMed Scopus (628) Google Scholar). The xanthophylls consist of two molecules of lutein (Lu), one neoxanthin (Nx), and one violaxanthin (Vx), bound to the sites L1/L2, N1, and V1, respectively. L1 and L2 are located close to the center of the complex, stabilizing a superhelix of protein helices I and III, whereas N1 and V1 are located more peripherally.The specificity of xanthophyll binding sites has been addressed both by analyzing xanthophyll biosynthetic mutants (see below) and by employing recombinant in vitro systems. In the latter approach, complexes were formed by having different carotenoids compete for the various binding sites. Lu and Nx were found to bind to the L1/L2 and N1 sites, respectively, with high specificity (3Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 4Hobe S. Niemeier H. Bender A. Paulsen H. Eur. J. Biochem. 2000; 267: 616-624Crossref PubMed Scopus (75) Google Scholar). As long as Lu is provided for binding to L1/L2, the N1 site does not bind any of the all-trans carotenoids Lu or Vx. Zx was shown to be a strong competitor for Lu, whereas Vx competes to a lesser extent. Consistently, when Lu is omitted completely from the refolding experiment, both Zx and Vx support complex formation by binding to L1/L2, although this increasingly compromises the complex stability. Moreover, Zx and Vx in L1/L2 compromise the binding specificity of N1, prompting its partial occupation with Vx. Xanthophyll biosynthetic mutants of Arabidopsis thaliana revealed a flexible response of the aggregational state of LHCIIb as well as of pigmentation of particular binding sites: double mutants lacking epoxycarotenoids as well as Lu exhibit a complete loss of trimeric LHCIIb with resulting monomeric complexes carrying only the remaining xanthophyll Zx (5Lokstein H. Tian L. Polle J.E.W. DellaPenna D. Biochim. Biophys. Acta - Bioenergetics. 2002; 1553: 309-319Crossref PubMed Scopus (145) Google Scholar, 6Tardy F. Havaux M. J. Photochem. Photobiol. B: Bio. 1996; 34: 87-94Crossref PubMed Scopus (75) Google Scholar). The loss of trimer formation and binding flexibility toward "non-native" carotenoids also holds true for single mutants where both the absence of Lu in mutant lut2, which is then replaced by Vx and/or Zx, and the absence of Nx in aba1 strongly decreases the trimer stability of LHCIIb (5Lokstein H. Tian L. Polle J.E.W. DellaPenna D. Biochim. Biophys. Acta - Bioenergetics. 2002; 1553: 309-319Crossref PubMed Scopus (145) Google Scholar, 6Tardy F. Havaux M. J. Photochem. Photobiol. B: Bio. 1996; 34: 87-94Crossref PubMed Scopus (75) Google Scholar, 7Hurry V. Anderson J.M. Chow W.S. Osmond C.B. Plant Phys. 1997; 113: 639-648Crossref PubMed Scopus (80) Google Scholar).The specificities of the L1, L2, and N1 binding sites appear to be based mostly on their selectivity for different polyene conformations, followed by a gradual response to different ionon ring types. In the case of L1/L2 a hydroxyl group at C3 on the β-end group turned out to be important (8Phillip D. Hobe S. Paulsen H. Molnar P. Hashimoto H. Young A.J. J. Biol. Chem. 2002; 277: 25160-25169Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In contrast to Lu and Vx, which both exhibit an all-trans configuration of the extended polyene chain, Nx in photosynthetic tissue of chlorophyll b-containing organisms is present in its 9′-cis conformation (9Takaichi S. Mimuro M. Plant Cell Phys. 1998; 39: 968-977Crossref Scopus (68) Google Scholar). However, pigment analysis of LHCIIb from the parasitic plant Cuscuta reflexa identified 9′-cis-Vx as the carotenoid in N1 (10Snyder A.M. Clark B.M. Robert B. Ruban A.V. Bungard R.A. J. Biol. Chem. 2004; 279: 5162-5168Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), suggesting that this binding site requires a 9-cis-5,6-epoxy-3-hydroxy carotenoid. This suggestion was confirmed by the x-ray structure of LHCIIb resolved at 2.72 Å (1Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1350) Google Scholar), which identified a tyrosine residue located in the luminal loop as the hydrogen bond partner for the ionone ring proximal to the 9′-cis conformation of Nx. Furthermore, numerous chlorophylls and amino acid side chains form a hydrophobic cleft that accommodates the bent polyene chain of Nx.Whereas both Lu and Nx are generally considered to be tightly bound to LHCIIb, the binding strength of Vx is much weaker. Solubilization of thylakoids usually causes the loss of Vx from LHCIIb unless special care is taken (11Ruban A.V. Lee P.J. Wentworth M. Young A.J. Horton P. J. Biol. Chem. 1999; 274: 10458-10465Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). X-ray crystallography localized the Vx binding site at the interface between two monomers in trimeric LHCIIb (1Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1350) Google Scholar, 2Standfuss J. Van Scheltinga A.C.T. Lamborghini M. Kühlbrandt W. EMBO J. 2005; 24: 919-928Crossref PubMed Scopus (628) Google Scholar) where the binding pocket is shaped by several amino acid side chains, chlorophylls, and lipids.Xanthophylls not only contribute to the structural stability of the pigment·protein complex but fulfill a number of additional tasks (12Horton P. Ruban A.V. Walters R.G. Annu. Rev. Plant Phys. Plant Mol. Biol. 1996; 47: 655-684Crossref PubMed Scopus (1398) Google Scholar, 13Niyogi K.K. Annu. Rev. Plant Phys. Plant Mol. Biol. 1999; 50: 333-359Crossref PubMed Scopus (1597) Google Scholar, 14Bassi R. Caffarri S. Photosynth. Res. 2000; 64: 243-256Crossref PubMed Scopus (142) Google Scholar, 15Müller P. Li X.P. Niyogi K.K. Plant Phys. 2001; 125: 1558-1566Crossref PubMed Scopus (2039) Google Scholar, 16Adamska I. Kruse E. Kloppstech K. J. Biol. Chem. 2001; 276: 8582-8587Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 17Holt N.E. Fleming G.R. Niyogi K.K. Biochem. 2004; 43: 8281-8289Crossref PubMed Scopus (267) Google Scholar). One of these is the protection against photodamage by quenching either the triplet-excited state of chlorophyll or the singlet state of oxygen. These two quenching processes have been assigned to Lu and Nx, respectively (3Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Another function of carotenoids is the dissipation of excess excitation energy in the light-harvesting apparatus involving zeaxanthin, generated by de-epoxidation of Vx in the xanthophyll cycle (12Horton P. Ruban A.V. Walters R.G. Annu. Rev. Plant Phys. Plant Mol. Biol. 1996; 47: 655-684Crossref PubMed Scopus (1398) Google Scholar, 13Niyogi K.K. Annu. Rev. Plant Phys. Plant Mol. Biol. 1999; 50: 333-359Crossref PubMed Scopus (1597) Google Scholar, 18Demmig-Adams B. Adams III W.W. Annu. Rev. Plant Phys. Plant Mol Biol. 1992; 43: 599-626Crossref Scopus (2044) Google Scholar). During this cycle, Vx presumably is extracted from its binding site in light-harvesting complexes to interact with the water-soluble de-epoxidase, which then reinserts its product back into the protein complex (19Jahns P. Wehner A. Paulsen H. Hobe S. J. Biol. Chem. 2001; 276: 22154-22159Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This dynamic binding behavior may explain why Vx easily dissociates from LHCIIb. The dynamics of carotenoid binding to LHCIIb is an issue to be considered when the biogenesis of this complex is to be understood. It is unclear how and in which sequence the carotenoids are assembled into the light-harvesting complexes. In an early stage of greening, before the massive accumulation of trimeric LHCIIb sets in, the monomeric complex is transiently found (20Dreyfuss B.W. Thornber J.P. Plant Phys. 1994; 106: 829-839Crossref PubMed Scopus (87) Google Scholar). However, no earlier assembly intermediate, containing only a subset of LHCIIb pigments, has ever been observed in greening plants. Of course this does not exclude the existence of such intermediates, because the biogenesis of LHCIIb is not well synchronized in greening tissue, and, therefore, such intermediates may appear only in low amounts at any given time point. Time-resolved fluorescence and CD measurements (21Booth P.J. Paulsen H. Biochem. 1996; 35: 5103-5108Crossref PubMed Scopus (73) Google Scholar, 22Horn R. Paulsen H. J. Biol. Chem. 2004; 279: 44400-44406Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) showed that the presence of Lu accelerates complex assembly and protein folding, more so than the presence of any other carotenoids, indicating that the correct occupation of the central xanthophyll binding sites L1 and L2 with Lu possibly precedes the binding of (some) other pigments (23Reinsberg D. Ottmann K. Booth P.J. Paulsen H. J. Mol. Biol. 2001; 308: 59-67Crossref PubMed Scopus (36) Google Scholar). In fact, LHCIIb containing Lu as the only carotenoid component is stable (4Hobe S. Niemeier H. Bender A. Paulsen H. Eur. J. Biochem. 2000; 267: 616-624Crossref PubMed Scopus (75) Google Scholar, 24Croce R. Remelli R. Varotto C. Breton J. Bassi R. FEBS Lett. 1999; 456: 1-6Crossref PubMed Scopus (104) Google Scholar), at least in its monomeric form (5Lokstein H. Tian L. Polle J.E.W. DellaPenna D. Biochim. Biophys. Acta - Bioenergetics. 2002; 1553: 309-319Crossref PubMed Scopus (145) Google Scholar). The present work addresses the question of whether Nx can bind to a potential intermediate of LHCIIb assembly consisting of the folded protein, Lu, and the chlorophylls. We show that in fact Nx binding to LHCIIb is reversible and, by using Nx-specific signals in the CD of LHCIIb, determine the binding constant and activation energy of its binding equilibrium.EXPERIMENTAL PROCEDURESPigment Preparation—9′-cis-Nx, all-trans-Vx, all-trans-Lu as well as chlorophylls a and b were prepared from pea leaves as described previously (21Booth P.J. Paulsen H. Biochem. 1996; 35: 5103-5108Crossref PubMed Scopus (73) Google Scholar). 9′-cis-Vx was extracted from papaya fruits (Carica sp., local market). The extraction of total pigments from papaya, the hydrolysis step in a KOH-ethanol solution, the extraction of carotenoids, and their fractionation by HPLC followed the procedure for carotenoids isolation from pea leaves (see above). 9′-cis-Vx was identified by the 40 nm hypsochromic shift in the absorption and increased hydrophobicity of its furanoid derivative produced by adding HCl (25Eugster C.H. Chemical derivatizations: Microscale tests for the presence of common functional groups in carotenoids.in: Britton G., L.-J.S. Pfender H. Carotenoids. Birkhäuser, Basel1995Google Scholar) and distinguished from the all-trans isomer by its slightly lower polarity and a 4–5 nm hypsochromic shift of absorbance (26Molnar P. Szabolcs J. Phytochem. 1980; 19: 623-627Crossref Scopus (33) Google Scholar).Preparation of Recombinant and Native LHCIIb—Native LHCIIb was isolated as described before (27Krupa Z. Huner N.P.A. Williams J.P. Maissan E. James D.R. Plant Physiol. 1987; 84: 19-24Crossref PubMed Google Scholar). Recombinant LHCIIb was prepared from overexpressed apoprotein carrying a His tag at its C terminus (28Rogl H. Kosemund K. Kühlbrandt W. Collinson I. FEBS Lett. 1998; 432: 21-26Crossref PubMed Scopus (162) Google Scholar). Monomeric samples were reconstituted by the detergent exchange method as described previously (29Paulsen H. Finkenzeller B. Kuhlein N. Eur. J. Biochem. 1993; 215: 809-816Crossref PubMed Scopus (164) Google Scholar) but without sucrose in the solubilization buffer to facilitate the subsequent purification step on sucrose gradients (0.1–1 m sucrose, 0.1% β-DM, 5 mm Tricine-NaOH, pH 7.8). Monomeric complexes were harvested with a syringe and kept on ice until use. Trimeric LHCIIb was prepared by detergent exchange reconstitution (29Paulsen H. Finkenzeller B. Kuhlein N. Eur. J. Biochem. 1993; 215: 809-816Crossref PubMed Scopus (164) Google Scholar) and affinity chromatography (30Yang C. Horn R. Paulsen H. Biochem. 2003; 42: 4527-4533Crossref PubMed Scopus (28) Google Scholar) and purified on sucrose gradients as described for monomeric complexes.Carotenoid Insertion Reactions—Insertion reactions were carried out with complexes extracted from sucrose gradients, thus containing ∼0.2 m sucrose, 0.1% β-DM, 5 mm Tricine-NaOH, pH 7.8. When unbound pigments were to be extracted upon the insertion reaction, sucrose was depleted from the complex solution. In this case, β-DM was added to give a total detergent concentration of 0.3%. The sample was then diluted 10-fold in 5 mm Tricine-NaOH, pH 7.8, and concentrated again on 30-kDa Centricon devices to the original volume, thereby decreasing the sucrose concentration to ∼0.02 m. Concentration of LHCIIb was determined using a molar extinction coefficient at 670 nm of 5.46 × 105 (m–1 × cm–1) (31Butler P. Kühlbrandt W. Proc. Nati. Acad. Sci. U. S. A. 1988; 85: 3797-3801Crossref PubMed Google Scholar). Xanthophylls for insertion were first completely solubilized in ethanol (concentration of ∼1–2 μg/μl). This solution was diluted 1:10 with 0.1% β-DM, 5 mm Tricine-NaOH, pH 7.8, sonified in a bath sonifier for 1 min, and filtrated through 0.2-μm sterile filters. Pigment concentration in the filtrate was determined by analytical HPLC after butanol extraction (32Martinson T.A. Plumley F.G. Anal. Biochem. 1995; 228: 123-130Crossref PubMed Scopus (50) Google Scholar). Insertion was started by mixing LHCIIb and xanthophyll solutions. The xanthophyll solution never exceeded 5% of the final volume to facilitate the comparison of LHCIIb CD spectra taken before and after the insertion reaction by using a J-810 spectropolarimeter (Jasco, Gross-Umstadt, Germany). The temperature during the insertion reaction was controlled by a thermostatted water bath.Determination of Binding Constant K—According to the law of mass action the binding constant K is defined as in Equation 1.K=[NLL·LHCIIb][Nx]×[LL·LHCIIb](Eq. 1) The ratio of NLL·LHCIIb/LL·LHCIIb was determined via CD478/492 calibration (see Fig. 9), whereas the portion of unbound Nx in equilibrium [Nx] can be expressed as the difference between total Nx minus Nx concentration bound to LHCIIb. Because of the stoichiometry of 1 Nx per apoprotein this can be calculated as in Equation 2.[Nx]=[Nx(total)]-[NLL·LHCIIb](Eq. 2) Total Nx was quantified by HPLC, whereas [NLL·LHCIIb] can be calculated by taking into account the ratio of NLL·LHCIIb/LL·LHCIIb as determined via CD478/492 calibration and the total concentration of LHCIIb as determined by A670 with a molar extinction coefficient of 5.46 × 105 (m–1 × cm–1) (31Butler P. Kühlbrandt W. Proc. Nati. Acad. Sci. U. S. A. 1988; 85: 3797-3801Crossref PubMed Google Scholar) (Equation 3).[NLL·LHCIIb]=[LHCIIb(total)][NLL·LHCIIb][LL·LHCIIb]×[NLL·LHCIIb][LL·LHCIIb](Eq. 3) By substituting [NLL·LHCIIb] in Equation 2 with Equation 3 and [Nx] in Equation 1 with Equation 2, K can be determined.TABLE 2Binding constant K of NxNx/LL·LHCIIbBinding constant Km-10.323.4 × 1060.327.8 × 1060.484.6 × 1060.481.0 × 1070.621.0 × 1070.629.7 × 106Mean value7.6 × 106 Open table in a new tab Determination of Rate Constants—After obtaining an initial CD spectrum (500–440 nm) of LL·LHCIIb at the respective temperature, Nx was added at a 0.5 m ratio with thorough mixing. During the insertion reaction spectra were taken every 60 s. The temperature was controlled by an implemented Peltier-type thermostat. Amplitudes of negative peaks at 478 nm and 492 nm from every spectrum were extracted, and [NLL·LHCIIb] was determined as described above. Rate constants k were determined by using Equation 4 for a second order reaction,[P]=[A]0[B]0(1-e([B]0-[A]0)kt)[A]0-[B]0e([B]0-[A]0)kt(Eq. 4) with [P] = [NLL·LHCIIb]; [A]0 and [B]0 are the concentrations of Nx and LL·LHCIIb, respectively, at the beginning of the reaction, k is the rate constant, and t = time (seconds). An iterative fit procedure was performed by minimizing the sum of square deviations at all time points.RESULTSDissociation of Nx from LHCIIb Depends on Temperature and Protein Concentration—As a first approach to further understand the dynamics of xanthophyll binding to LHCIIb we tested whether Nx and Lu, like Vx, are reversibly bound, i.e. whether these pigments are able to dissociate from the otherwise intact complex. Native trimeric LHCIIb was first diluted in detergent buffer containing 0.1% β-DM. Afterward potentially dissociating pigments were separated from the remaining complexes by ultracentrifugation at different temperatures to overcome possible activation barriers of carotenoids dissociation. Fig. 1 depicts the relative pigment compositions of native LHCIIb as a result of both decreasing concentration and increasing temperature during isolation by sucrose density centrifugation. The complex concentrations given in Fig. 1 were measured before ultracentrifugation but are expressed as per gradient volume. The combination of nearly hundredfold dilution before centrifugation with a temperature increase up to 40 °C during preparative ultracentrifugation leads to a specific and complete loss of Nx but not Lu (Fig. 1). The dissociation of Nx from LHCIIb can both be observed with respect to Lu (Fig. 1A) and chlorophyll a plus b (Fig. 1B). Pigment compositions of the remaining complexes after Nx dissociation do not significantly change, as indicated by the fact that the graphs in Fig. 1 (A and B) follow the same profile.FIGURE 1A combination of temperature increase and dilution leads to specific loss of neoxanthin. Native trimeric LHCIIb was diluted with 0.1% β-DM and purified by sucrose density ultracentrifugation at the temperatures given below. Initial LHCIIb concentrations, determined by the absorbance at 670 nm, were measured before ultracentrifugation. Trimeric complexes were harvested, and their pigment contents analyzed by HPLC. A, Nx/Lu ratios; B, Nx/(Chla+b) ratios. Temperature during centrifugation (16 h) was as follows: squares, 4 °C; diamonds, 24 °C; triangles, 35 °C; and crosses, 40 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nx Rebinds at a Defined Stoichiometry and Correct Orientation—If this specific loss of Nx is based on an equilibrium reaction we would expect the remaining LL·LHCIIb to rebind Nx either upon reconcentrating the sample or upon adding an excess of unbound Nx. Because samples solubilized in a detergent system are hard to concentrate without changing the concentration of detergent, we chose the second approach. However, because biochemical and spectroscopic analysis of emerging complexes demand higher concentrations than that available after extreme dilution, we chose recombinant LHCIIb refolded in the absence of Nx as the substrate for Nx binding. In the presence of chlorophylls a and b and of Lu as the sole carotenoid, both monomeric and trimeric LHCIIb could be isolated by sucrose density ultracentrifugation (Fig. 2). Although the yield of trimeric complexes decreases when Nx is omitted from the refolding mixture, the composition of chlorophylls and remaining Lu molecules within the resulting monomers and trimers remained largely unchanged (Table 1). These complexes were then incubated with Nx solubilized in detergent and subsequently re-isolated on sucrose gradients. HPLC analysis of pigment composition of emerging complexes shows that both monomeric and trimeric LL·LHCIIb take up Nx in a stoichiometric manner (Table 1). This becomes obvious from the upper limit of one molecule of Nx per apoprotein, even under conditions of 3- and 5-fold excess over LL·LHCIIb, indicating Nx binding to be quite specific.FIGURE 2Preparative ultracentrifugation of monomeric and trimeric LL·LHCIIb and NLL·LHCIIb. Complexes were reconstituted by detergent exchange, trimerized on a nickel column, and separated over night by sucrose density ultracentrifugation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Xanthophyll composition of recombinant monomeric and trimeric LL·LHCIIb upon Nx insertionLHCIIb monomersLHCIIb trimersNxLuNxLumControlsNLL·LHCIIb1.01.91.01.9LL·LHCIIb2.22.3Deployed ratio of Nx/LL·LHCIIbInsertions0.330.72.10.50.62.03.01.02.15.01.01.9 Open table in a new tab To confirm this we also compared circular dichroic features, which are a sensitive tool for detecting pigment-pigment interactions. The CD spectra taken from NLL·LHCIIb and LL·LHCIIb in their monomeric states reveal significant differences both in the blue (400–500 nm) and the red (650–700 nm) regions of complex absorption (Fig. 3). In the red region the absence of Nx decreases CD amplitudes at 650 nm and the positive signal at 668 nm, whereas the signal strength at 681 nm is increased. In the Soret region we observed a stronger negative CD signal at 478 nm, and additionally, the amplitude of the negative CD signal at 492 nm was decreased. These signals presumably contain contributions of Nx interacting with chlorophyll b as suggested by the wavelength region and the spatial vicinity of binding site N1 and several chlorophylls b close to helix C (1Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1350) Google Scholar, 24Croce R. Remelli R. Varotto C. Breton J. Bassi R. FEBS Lett. 1999; 456: 1-6Crossref PubMed Scopus (104) Google Scholar, 33Palacios M.A. Caffarri S. Bassi R. Van Grondelle R. Van Amerongen H. Biochim. Biophys. Acta - Bioenergetics. 2004; 1656: 177-188Crossref PubMed Scopus (11) Google Scholar). Insertion of Nx transforms CD signals of LL·LHCIIb into those characteristic for NLL·LHCIIb as can be seen by comparing the spectra of NLL·LHCIIb and LL·LHCIIb with substoichiometric insertion of Nx into LL·LHCIIb (Fig. 4). Increasing ratios of Nx/LL·LHCIIb, offered successively, shift the Soret CD profile toward that of fully pigmented LHCIIb. This further corroborated the notion of a correct three-dimensional orientation of inserted Nx, as the CD characteristics become indistinguishable from native LHCIIb monomers (34Hobe S. Prytulla S. Kühlbrandt W. Paulsen H. EMBO J. 1994; 13: 3423-3429Crossref PubMed Scopus (156) Google Scholar).FIGURE 3CD spectra of monomeric LHCIIb with or without Nx. Upper panel: solid line, monomeric LL·LHCIIb; dashed line, fully pigmented monomeric NLL·LHCIIb. Lower panel: difference spectrum NLL·LHCIIb – LL·LHCIIb. Samples were diluted to the same optical density at 670 nm prior to CD measurement.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Nx insertion into LL·LHCIIb restores NLL·LHCIIb-specific CD signals. CD spectra were taken after Nx was inserted into LL·LHCIIb for 14 h at 0 °C at the following molar ratios of Nx/LL·LHCIIb. Squares, 0.32; circles, 0.48; and triangles, 0.62. CD spectra of LL·LHCIIb (dashed line) and NLL·LHCIIb (solid line) are given as references. Spectra were normalized to the isosbestic point (see Fig. 9) at 451 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Rebound Nx Is Functional—It was shown before that LL·LHCIIb exhibits a decreased fluorescence yield compared with fully pigmented NLL·LHCIIb (35Formaggio E. Cinque G. Bassi R. J. Mol. Biol. 2001; 314: 1157-1166Crossref PubMed Scopus (135) Google Scholar). Consistently with this observation, fluorescence rises upon association of Nx to prefolded LL·LHCIIb as monitored by comparing excitation spectra (Fig. 5). Fluorescence emission before (dashed line) and after (solid line) Nx insertion was measured at 720 nm, beyond the emission maximum, to be able to record an excitation spectrum covering the whole red region of chlorophyll absorption. Close inspection of the Soret region of excitation reveals a contribution of newly inserted Nx at ∼492 nm. This shoulder was also present in the spectrum of fully pigmented NLL·LHCIIb obtained by folding the protein in the presence of both Lu and Nx (Fig. 5, dotted line). The insertion reaction was allowed to proceed for 1 h at 12°C before taking the excitation spectra. The slight difference in the blue region between insertion product and fully pigmented NLL·LHCIIb did not indicate deficient coupling of Nx but was caused by Nx insertion not yet being completed after 1 h under these conditions (see below, Fig. 12).FIGURE 5Inserted Nx contributes to chlorophyll fluorescence and increases overall quantum yield. Excitation spectra of a LL·LHCIIb sample before (dashed line) and after (solid line) Nx insertion are compared. Insertion was carried out at 12 °C for 60 min. The dotted line represents a reference excitation spectrum of NLL·LHCIIb. This spectrum has been normalized to that of the Nx insertion experiment at their maximum signal in the red spectral domain where Nx does not contribute.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 12A, temperature-dependent formation of NLL·LHCIIb by insertion of Nx into LL·LHCIIb. Nx-free monomeric LL·LHCIIb was incubated with a 2-fold excess of Nx at the following temperatures, leading to the given k value (m–1 s–1); the minimum and ma
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