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J Biol Chem, Vol. 275, Issue 13, 9087-9090, March 31, 2000

ACCELERATED PUBLICATION
Chlorophyll Binding to Peptide Maquettes Containing a Retention Motif^*

Laura L. Eggink and J. Kenneth Hoober

From the Department of Plant Biology and The Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1601

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The motif Glu-X-X-His/Asn-X-Arg is conserved in the first and third membrane-spanning domains of all light-harvesting chlorophyll a/b- and a/c-binding proteins in chloroplasts. Molecular modeling of synthetic peptides containing the sequence Glu-Ile-Val-His-Ser-Arg, a motif found in the apoprotein of the major light-harvesting complex in plants, generated a loop structure formed by intrapeptide, electrostatic attraction between Glu and Arg. His, Asn, and charge-compensated Glu-Arg pairs are known ligands of the magnesium atom in chlorophyll. The prediction that this structure should bind two molecules of chlorophyll was confirmed experimentally with an assay based on fluorescence resonance energy transfer between peptides and chlorophyll a. Motifs with both potential ligands bound approximately two times the amount of chlorophyll as one in which His was replaced by Ala. These results support the conclusion that formation of this intermediate, within membranes of the envelope, is a crucial step in assembly of light-harvesting complexes and a mechanism that regulates import of the apoproteins into the chloroplast.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Most of the light energy that drives photosynthesis in plants is absorbed by Chl¹ a/b-containing light-harvesting complexes within thylakoid membranes. Apoproteins (Lhcb) of these complexes, encoded in the nuclear genome, are imported into chloroplasts after synthesis on ribosomes in the cytosol. Studies with the alga Chlamydomonas reinhardtii showed that completion of import of Lhcb by chloroplasts in vivo requires interaction with Chl (1). In vitro reconstitution of the complex with purified components Lhcb, Chl a, Chl b, and xanthophylls, such as lutein, violaxanthin, and/or neoxanthin, has been remarkably successful in identifying binding sites for Chl within the protein (2, 3). These studies have also shown that binding of Chl induces folding of Lhcb (4). However, with the complete protein, the initial event in assembly of LHCII is shrouded by the myriad interactions that occur during reconstitution. To reduce analysis of the interaction of Chl with the protein to its simplest, most fundamental level, we adopted a minimalist approach with short (16-mer), synthetic peptides, designated maquettes (5, 6). We focused on the amino acid sequence in the first membrane-spanning domain (helix-1) of Lhcb, which contains specific residues identified by mutagenesis as critical for import into the chloroplast (7-9) and for reconstitution of LHCII (3, 10). In particular, the motif EXXHXR, or less commonly EXXNXR, is conserved in helix-1 of all apoproteins that bind Chl a and Chl b, whether specific for photosystem I (Lhca) or photosystem II (Lhcb) (11). The third membrane-spanning domain of these proteins also contains this motif, with EXXNXR as the predominant sequence. Evidence of remarkable conservation of the motif throughout evolution is the occurrence of these motifs in apoproteins of light-harvesting complexes containing Chl a and Chl c in chromophytic algae and dinoflagellates (12) and in small Lhcb-like homologs in cyanobacteria (13). The critical amino acids correspond to residues Glu⁶⁵, His⁶⁸, and Arg⁷⁰ in the sequence of pea Lhcb1 (14). (Other amino acids in this sequence are conserved to a lesser extent and indicated as "X" to highlight the functional residues). His or Asn residues in proteins are common ligands of the magnesium atom in Chl, and Glu in an ion pair with Arg also functions as a ligand in LHCII (14). The sequence was designated a "retention" motif in recognition of its proposed role in retaining the protein within the chloroplast during import (1). In this paper we describe binding of Chl to the motif, an interaction that is expected to initiate LHCII assembly and thereby determine whether the apoprotein is retained in the chloroplast.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Preparation of Maquettes and Chl-- Peptides were synthesized in the Protein Chemistry Facility at Arizona State University on a Milligen model 9050 Plus continuous flow peptide synthesizer using Fmoc (9-fluorenylmethoxycarbonyl)-protected amino acids. Each product was purified by reverse-phase HPLC and checked for purity and correct molecular weight by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Chl was extracted with acetone from light-grown C. reinhardtii cw15 cells, and Chl a was purified by HPLC on a semipreparative ODS-C₁₈ reverse-phase column eluted at room temperature with 100% methanol as described previously (15). Fractions were evaporated to dryness under a stream of nitrogen and Chl was dissolved in a small volume of ethanol. Concentration of Chl was determined with published extinction coefficients (16).

Assay Conditions and Spectroscopy-- The assay routinely included 200 nM Chl a and various concentrations of peptide in 50 mM sodium borate, pH 9.0, containing 30 mM n-octyl--D-glucopyranoside (OG) or 1 mM n-dodecyl--D-maltoside (DM). The mixture was incubated at 36 °C in the dark until a steady-state level of fluorescence was achieved, which was usually 15 min. Fully corrected excitation spectra were obtained from 250 nm to 500 nm by measuring emission at 675 nm with a FluoroMax spectrofluorometer (Spex Industries, Edison, NJ). A 600-nm cut-off filter was placed in front of the emission spectrometer to eliminate secondary excitation peaks. CD spectra were recorded at room temperature with a Jasco-710 spectropolarimeter utilizing J-700 software, with a cell path length of 0.1 cm, in the continuous mode by taking points every 0.1 nm. Scan rate was 50 nm/min with 1-nm bandwidth and integration time of 1 s.

Molecular Modeling-- Model building, energy minimization, and molecular dynamics were conducted using INSIGHT II version 97.2 BUILDER, BIOPOLYMER, and DISCOVER modules (Molecular Simulations Inc., San Diego, CA). The consistent valence force field was used in all simulations and dynamics calculations were performed at 300 K. Structural simulations were initiated with the peptide in the fully extended form.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Maquettes were synthesized with the simplified structures shown in Fig. 1 to reduce the influence of amino acid residues adjacent to the motif (-EIVHSR-). Molecular modeling predicted formation of an intramolecular loop, by electrostatic pairing of Glu and Arg, from which the side chain of His protruded. Consequently, the peptide should contain two ligands that interact with Chl (Fig. 2). The Trp adjacent to the motif in the primary sequence of Lhcb1 (Fig. 1) was included to provide an assay for the interaction. Orientation of the transition dipole of the Trp side chain should favor fluorescence resonance energy transfer (FRET) to any Chl molecules bound to the peptide. The fluorescence emission maximum of Trp overlaps maxima at 338 and 378 nm in the absorption spectrum of Chl a, as required for FRET (17). In addition, absorbance of Trp allowed quantitation of the peptide. Our strategy was to mix Chl with maquettes in a buffer containing a detergent at concentrations above the critical micelle concentration (CMC) to achieve a nonpolar environment analogous to a membrane.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Sequences of maquettes. The upper line is the sequence of amino acids in the first membrane-spanning region of pea Lhcb1, positions 60-75. The N- and C-terminal segments of the maquette and inverted peptides were modified from the native sequence to remove influence from polar amino acid side chains adjacent to the potential Chl-binding motif. Large type indicates the proposed functional amino acids in the motif.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   A model of the proposed complex formed between the maquette and Chl. The model shows the initial secondary structure that formed during molecular dynamics of the extended peptide. The N terminus is toward the top of the figure. The Chl molecules were positioned near the ligands in the loop formed by the Glu/Arg ion pair, shown to the left, and His to the right, of the peptide. Side chain atoms of Glu, Arg, and His residues are highlighted in color: oxygen, red; nitrogen, blue; and carbon, green. The adjacent Trp side chain is shown in yellow. Chl molecules are shown in green, with magnesium in magenta.

OG (CMC, 25 mM) was not sufficiently nonpolar to prevent aggregation of Chl, and thus fluorescence was strongly quenched (Fig. 3A). After addition of the maquette to this mixture, an excitation spectrum emerged that was typical of Chl a between 300 and 450 nm, but which also included a strong maximum at 280 nm, the absorption maximum of Trp. However, a control peptide in which His and Glu were replaced with Ala (see Fig. 1) was also able to produce an excitation spectrum similar to that shown in Fig. 3A, which indicated that in this system the peptides bound Chl nonspecifically. It is notable that the ratio of emission from excitation at 280 nm relative to that at 434 nm, the Soret maximum of Chl a, remained constant as the concentration of peptide increased, which suggested that each peptide extracted a defined number of Chl molecules from the aggregate.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   FRET analysis of binding of Chl to the maquette. Excitation spectra were obtained between 250 and 500 nm, with emission measured at 675 nm. A, spectra of the mixture of 200 nM Chl a with the maquette in buffer containing 30 mM OG: trace a, no peptide; trace b, 10 µM and trace c, 20 µM maquette. B, spectra of the reaction of 200 nM Chl a in buffer containing 1 mM DM, with the peptides added in 6 M urea: trace a, no peptide; trace b, 20 µM maquette; trace c, 40 µM maquette; and trace d, 20 µM control peptide with Glu and His replaced with Ala.

The fluorescence yield of Chl in 1 mM DM (CMC, 0.17 mM) was several orders of magnitude greater than that in OG, which indicated that Chl was dispersed into the larger, more nonpolar micelles of DM. An excitation maximum at 280 nm was not detected when the maquette was added to this mixture, and the fluorescence emission maximum of Trp in the peptide remained near 356 nm, indicating that it was excluded from the nonpolar environment. With the possibility that the peptide assumed a secondary structure that prevented penetration of the micelle, the peptide was dissolved in 6 M urea and subsequently diluted into the assay mixture. As shown in Fig. 3B, excitation spectra for this mixture included a maximum at 280 nm that was not present in the spectrum of Chl alone. The maximum at 280 nm was not detected when the control peptide, in which both ligands were eliminated by replacing Glu and His with Ala, was added in urea. Thus, binding of Chl to the maquette in this mixture was sequence-dependent. Higher concentrations of urea in the assay mixture diminished the maximum in the excitation spectra at 280 nm, which suggested that urea served as a competing ligand and prevented Chl-peptide interaction. Thus we took another approach to study this process.

Peptides with inverted sequence often have different physical properties (18). To test whether an inverted maquette would bind Chl, the 16-mer peptide was synthesized in reverse order to provide the motif as -RSHIVE- in the N- to C-terminal direction. Molecular modeling predicted a loop structure in the inverted maquette similar to that shown in Fig. 2 for the forward sequence. The inverted peptide was more soluble in water yet incorporated readily into the nonpolar environment of DM micelles, as indicated by a blue shift in the emission maximum of W from 356 nm for the peptide in buffer to about 330 nm in the buffer-DM mixture (not shown). Fluorescence of Chl, from a maximum at 280 nm in the excitation spectrum, increased dramatically as the concentration of peptide increased, reaching a level greater than that with direct excitation of Chl at 434 nm (Fig. 4A). The emission spectrum of Chl did not change as peptide was added, except for the increase in intensity (Fig. 4B). The excitation difference spectrum between no peptide and 5 µM peptide, from 250 to 300 nm, was characteristic of the absorption spectrum of Trp (Fig. 4C). The lack of significant structure in the difference spectrum from 320 to 450 nm indicated that direct excitation of Chl was not altered by addition of peptide.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   FRET analysis of binding of Chl to inverted maquettes in DM micelles. A, excitation spectra of mixtures of Chl and no peptide (trace a) or 2 µM (trace b), 5 µM (trace c), and 10 µM (trace d) peptide. B, emission spectra of Chl a with no peptide, excited at 434 nm (trace a) or plus 10 µM inverted maquette, excited at 280 nm (trace b). C, difference excitation spectrum between 200 nM Chl with no peptide and with 5 µM inverted maquette. D, graphical presentation of the intensity of fluorescence at 675 nm with excitation at 280 nm, relative to that at 434 nm, as a function of the concentration of peptide: , inverted maquette; , single-ligand, inverted maquette in which His was replaced with Ala; and , control peptide in which His and Glu were replaced with Ala. E, double-reciprocal plots of data in D for inverted maquettes with two () or one () ligand. F, far-UV CD spectra of peptides: trace a, inverted maquette; trace b, control peptide with His and Glu replaced with Ala; trace c, maquette; and trace d, single-ligand, inverted maquette with His replaced with Ala. Spectra were determined at equal concentrations (100 µM) of peptide in 50 mM sodium borate, pH 9.0, containing 1 mM DM.

Two additional peptides were tested, one with the inverted motif modified to a single ligand by replacement of His with Ala (-RSAIVE-) and the control peptide in which His and Glu were replaced with Ala (-RSAIVA-) (see Fig. 1). Graphical analysis of the interaction of Chl with these peptides, expressed as a ratio of peptide-enhanced emission by excitation at 280 nm relative to excitation of Chl at 434 nm, yielded typical binding curves (Fig. 4D). The increase in fluorescence at 675 nm by energy transfer from the single-ligand peptide was approximately half that with the two-ligand peptide at the same concentration. The slopes as well as the y intercepts of double-reciprocal plots of these data (Fig. 4E) indicated that the peptide with two ligands bound approximately two times more Chl than that with a single ligand. The lack of energy transfer from Trp to Chl with the peptide that did not contain a ligand (Fig. 4D) confirmed that Chl binding to the maquettes under these conditions was sequence dependent. An experiment with free Trp showed that a concentration 1000-fold greater than that of the peptides was required to detect fortuitous energy transfer between the amino acid and Chl (not shown). Because hydroxyl groups serve as ligands to Chl (19), we tested whether ethanol, the solvent in which Chl was added to the assay, interfered with binding to the peptide. Increasing the concentration of ethanol to 200 µM, a concentration 5-fold greater than typically in the assay, slightly increased FRET but higher concentrations caused a decrease (not shown). The small effect of such high concentrations of ethanol relative to that of peptide indicated that the alcohol did not significantly affect the binding results. This result also suggests that the hydroxyl group of the serine residue in the retention motif does not have significant ligand activity.

These data show that Chl interacts with the motif in either orientation. Concentrations of peptides that enhanced fluorescence were in the range of 5-50-fold greater than that of Chl. Either the affinity of maquettes for Chl was low or the experimental conditions did not favor interaction. In support of the latter possibility, the number of micelles in the assay mixture, estimated assuming 80 detergent molecules per micelle (Anatrace, Inc.), exceeded the number of Chl molecules approximately 50-fold. The number of micelles was nearly equal to that of peptide molecules, although from the characteristics of fluorescence of Trp, incorporation of peptide into micelles was not quantitative. Thus, equilibration into, and interaction between, micelles may have been required to achieve interaction of a peptide with more than one molecule of Chl. Once formed, the complex seemed relatively stable, because no loss of FRET was detected within an hour after a 1000-fold dilution of reactants into the same buffer-detergent mixture. The greater emission of Chl as a result of energy transfer from a peptide (Fig. 4, A and D) suggested that FRET increased fluorescence yield. Fluorescence intensity of Chl at the same concentration in acetone was 10-fold greater than that in the assay mixture (not shown), which indicated that the yield in the latter environment was relatively low.

Far-UV CD spectra showed that the secondary structures of the peptides varied substantially. The inverted maquette existed predominantly as a random coil in buffer containing DM (Fig. 4F, trace a), a conformation that apparently facilitated interaction with Chl. Ion pair formation between the positively and negatively charged Arg and Glu residues spaced i + 5 apart is greater than the helix-stabilizing distance of i + 4 (18) and is expected to form a loop that prevents helix formation. Also, the Arg-Glu orientation in the inverted sequence should interact less favorably with the helix dipole than the Glu-Arg orientation and thus be a helix-destabilizing factor (18). When Glu in the retention motif was replaced with Ala, thereby eliminating the ion pair, the peptide was largely -helical (Fig. 4F, trace b). The spectrum of the forward maquette (Fig. 4F, trace c) was characteristic of a random coil plus a -hairpin. Support for the latter assignment was the spectrum of the single-ligand peptide, with His replaced by Ala (Fig. 4F, trace d), which had a weak signal characteristic of -sheet structure generated by a -turn (21, 22). These spectra support formation of the loop structure as proposed by the model in Fig. 2 for the maquette.

These experiments provide direct evidence that such motifs in apoproteins of light-harvesting complexes bind Chl molecules, consistent with the predicted function of these sequences in Lhcb (1). The environment within the chloroplast envelope probably influences their secondary structure as Lhcb precursors are transferred through the import apparatus. Removal of the transit sequence from precursors occurs rapidly (20, 23), and when the capacity for Chl synthesis is sufficiently high, interaction with newly synthesized Chl locks the proteins into the surrounding membrane. The fact that mature-sized Lhcb does not accumulate in the chloroplast in vivo when Chl is not available for assembly of LHCII, but instead accumulates in the cytosol or in vacuoles (20), convincingly demonstrated that interaction with Chl must occur as apoproteins pass into the chloroplast envelope. The reduced level of binding of Chl by peptides containing a single ligand provides an explanation for the low accumulation in chloroplasts of mutant forms of Lhcb1 that lack one of the ligands (7, 8). Chl seems to be the limiting factor for LHCII assembly (20, 23), and therefore association of two Chl molecules with the native motif is expected to provide a powerful mechanism that regulates entry of Lhcb into the chloroplast.

	FOOTNOTES

^* This work was supported by Graduate Training Grant DGE9553456 from the National Science Foundation. This is publication number 423 from the Center for the Study of Early Events in Photosynthesis.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Plant Biology, Arizona State University, Tempe, AZ 85287-1601. Tel.: 480-965-3414; Fax: 480-965-6899; E-mail: khoober@asu.edu.

	ABBREVIATIONS

The abbreviations used are: Chl, chlorophyll; CMC, critical micelle concentration; DM, n-dodecyl--D-maltopyranoside; OG, n-octyl--D-glucopyranoside; FRET, fluorescence resonance energy transfer; HPLC, high-pressure liquid chromatography; Lhca and Lhcb, apoproteins of light-havesting complexes associated with photosystem I or II, respectively; LHCII, light-harvesting complex associated primarily with photosystem II.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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