Article pubs.acs.org/biochemistry
Reversible Changes in the Structural Features of Photosynthetic Light-Harvesting Complex 2 by Removal and Reconstitution of B800 Bacteriochlorophyll a Pigments Yoshitaka Saga,*,†,‡ Keiya Hirota,† Hitoshi Asakawa,‡,§,∥ Kazufumi Takao,§ and Takeshi Fukuma§,∥ †
Department of Chemistry, Faculty of Science and Engineering, Kindai University, Higashi-Osaka, Osaka 577-8502, Japan Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan § Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan ∥ Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa 920-1192, Japan ‡
S Supporting Information *
ABSTRACT: Light-harvesting complex 2 (LH2) is an integral membrane protein in purple photosynthetic bacteria. This protein possesses two types of bacteriochlorophyll (BChl) a, termed B800 and B850, which exhibit lowest-energy absorption bands (Qy bands) around 800 and 850 nm. These BChl a pigments in the LH2 protein play crucial roles not only in photosynthetic functions but also in folding and maintaining its protein structure. We report herein the reversible structural changes in the LH2 protein derived from a purple photosynthetic bacterium, Rhodoblastus acidophilus, induced by the removal of B800 BChl a (denoted as B800-free LH2) and the reconstitution of exogenous BChl a. Atomic force microscopy observation clearly visualized the nonameric ring structure of the B800-free LH2 with almost the same diameter as the native LH2. Size exclusion chromatography measurements indicated a considerable decrease in the size of the protein induced by the removal of B800 BChl a. The protein size was almost recovered by the insertion of BChl a pigments into the B800 binding sites. The decrease in the LH2 size would mainly originate from the shrinkage of the B800 binding sites perpendicular to the macrocycle of B800 BChl a without deformation of the circular arrangement. The reversible changes in the LH2 structure induced by the removal and reconstitution of B800 BChl a will be helpful for understanding the structural principle and the folding mechanism of photosynthetic pigment−protein complexes.
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hotosynthetic light-harvesting proteins consist of polypeptides and photofunctional pigments, such as chlorophyll, bacteriochlorophyll (BChl), and carotenoid molecules. The pigments are highly organized in the scaffold of the polypeptide assemblies, resulting in the efficient capture and transfer of solar energy. The architecture of photosynthetic light-harvesting proteins has attracted considerable attention in diverse areas such as photobiology, protein science, and artificial photosynthesis.1−5 Light-harvesting complex 2 (LH2) in purple photosynthetic bacteria is one of the most famous photoactive proteins in nature because of its beautiful structure and photofunctions. BChl a pigments are regularly arranged in a circular form in this protein. The three-dimensional (3D) structures of the LH2 proteins derived from two purple photosynthetic bacteria, Rhodoblastus (Rbl.) acidophilus6,7 and Phaeospirillum (Phs.) molischianum,8 have been elucidated at high resolution; the LH2 proteins from the former and latter species have C9 and C8 symmetries, respectively (the structure of LH2 from Rbl. acidophilus is shown in Figure 1). BChl a pigments in the LH2 © XXXX American Chemical Society
Figure 1. Structure of the LH2 protein from Rbl. acidophilus (Protein Data Bank entry 1NKZ): left, top view; right, side view. B800 and B850 BChl a are colored magenta and green, respectively.
Received: March 22, 2017 Revised: May 26, 2017
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DOI: 10.1021/acs.biochem.7b00267 Biochemistry XXXX, XXX, XXX−XXX
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(Rba.) sphaeroides 2.4.1 and purified via reverse-phase highperformance liquid chromatography according to previous reports.26,27 A methanol solution of BChl a (0.2 mM) was mixed with a solution of B800-free LH2 (0.16 μM) in 20 mM Tris-HCl buffer containing 0.1% DDM (pH 4.5). The volume ratio of the BChl a solution and the B800-free LH2 solution was 1:19. Then, the pH of the solution was adjusted to pH 8.0, followed by incubation at 35 °C for 2 h. The sample was concentrated via ultrafiltration using Amicon centricon concentrators (30 kDa cutoff) and loaded onto a SephacrylS200 column in 20 mM Tris-HCl buffer containing 0.1% DDM and 150 mM NaCl (pH 8.0). The protein collected was desalted by ultrafiltration using Amicon centricon concentrators (30 kDa cutoff). Spectroscopic Measurements. Electronic absorption and circular dichroism (CD) spectra of LH2 proteins were measured with a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) and a JASCO J-820 spectropolarimeter (JASCO, Tokyo, Japan), respectively. Fluorescence emission spectra were measured with a Hamamatsu C992003G fluorescence measurement system (Hamamatsu Photonics, Shizuoka, Japan). Apparent fluorescence quantum yields of B850 BChl a were calculated from the emission above 840 nm by software installed in a Hamamatsu C9920-03G fluorescence measurement system. Size Exclusion Chromatography. LH2 proteins were analyzed by SEC on a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare) with a mixed buffer of 20 mM Tris and 10 mM succinate containing 0.1% DDM and 150 mM NaCl at a flow rate of 0.4 mL min−1. pH Titration of Simultaneous Dynamic Light Scattering and Electronic Absorption Spectroscopy. A native LH2 protein was stirred in 20 mM Tris-HCl buffer containing 0.1% DDM at each pH, which was controlled by HCl and aqueous KOH, followed by the measurements of electronic absorption spectra and hydrodynamic diameters with a Shimadzu UV-2450 spectrophotometer (Shimadzu) and a Malvern Zetasizer Nano-ZSP particle size analyzer (Malvern Instruments, Malvern, U.K.), respectively. These experiments were performed in the pH range of 3−8. AFM Measurements. A 100 μL portion of a solubilized LH2 solution in 20 mM Tris-HCl buffer containing 0.1% DDM and 150 mM NaCl (pH 8.0 and 5.0 for native/reconstituted LH2 and B800-free LH2, respectively) was deposited on a cleaved mica substrate with a diameter of 12 mm (SPI Supplies, West Chester, PA), followed by standing for 30 min at room temperature in the dark. Then, the sample was rigorously rinsed with 20 mM Tris-HCl buffer containing 150 mM NaCl at the corresponding pH. The sample was observed in 20 mM TrisHCl buffer containing 150 mM NaCl at the corresponding pH by a home-built frequency modulation AFM (FM-AFM) instrument with a silicone cantilever (PPP-NCHAuD, Nanoworld AG, Neuchâtel, Switzerland), which had a nominal spring constant of 42 N/m. The typical resonance frequency and Q factor in an aqueous buffer solution are 150 kHz and 7, respectively.28,29 A commercially available AFM controller (ARC2, Asylum Research, Santa Barbara, CA) was used to control the FM-AFM system.28,29
protein are classified into two types: B800 and B850 BChl a. B800 BChl a is present as a monomeric form in the protein scaffold and exhibits the lowest-energy absorption band (Qy band) around 800 nm. In contrast, B850 BChl a pigments are assembled as a dimeric form in one unit and interact excitonically in the dimer as well as between dimers in a circular arrangement. The light energy captured by B800 BChl a is efficiently transferred to B850 BChl a in the LH2 proteins. Chlorophyllous pigments bound to photosynthetic proteins play important roles not only in the photofunction of these proteins but also in folding and maintaining the protein structures. B800 BChl a has been recognized as a good structural probe of LH2 proteins,9−18 because its spectral features are sensitive to the local protein structure. The spectral changes in B800 BChl a have so far been monitored by various measurements, such as electronic absorption spectroscopy under high-pressure9−12 and single-molecule spectroscopy,13−16 to obtain structural information about the proximity of the B800 binding sites in LH2 proteins. Alternatively, exogenous (B)Chl pigments have been reconstituted into LH2 lacking B800 BChl a to study interactions with amino acid residues that form B800 binding sites and energy transfer mechanisms from B800 to B850 BChl a.19−23 Cogdell, Scheer, and co-workers performed a pioneering work in this research area.20 In spite of these excellent investigations of chlorophyllous pigments that are bound to the B800 binding sites in the LH2 protein, however, little information is available about the overall structure and properties of LH2 without B800 BChl a (hereafter denoted B800-free LH2 protein). The physicochemical features of the B800-free LH2 protein will provide new insight into the architecture and functions of LH2, although scant attention has so far been paid to the B800-free LH2 itself. In this study, we report the spectral and structural properties of the B800-free LH2 protein prepared from a purple photosynthetic bacterium, Rbl. acidophilus, and a protein that is reconstituted with exogenous BChl a into empty B800 binding sites (denoted as reconstituted LH2) by spectroscopic measurements, size exclusion chromatography (SEC), and atomic force microscopy (AFM).
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MATERIALS AND METHODS Preparation of Native and B800-Free LH2 Proteins. A native LH2 protein was isolated from the cultured cells of Rbl. acidophilus DSM137 (formerly known as Rhodopseudomonas acidophila 7050, the type strain of Rbl. acidophilus24) according to a previous report.25 The isolated LH2 protein was solubilized in 20 mM Tris-HCl buffer containing 0.1% n-dodecyl β-Dmaltoside (DDM) (pH 8.0), in which the B850 absorbance was adjusted to 0.5. Then, an aliquot of 10% Triton X-100 was added to this solution (the final concentration of Triton X-100 was 0.05%), and the pH of this solution was adjusted to 4.0 with 10% acetic acid, followed by incubation at 35 °C for 1 h. The detergents DDM and Triton X-100 were purchased from Dojindo Laboratories, Co. (Kumamoto, Japan), and Wako Chemical Industry, Co. Ltd. (Osaka, Japan). The B800-free LH2 protein obtained was purified via cation-exchange column chromatography using a Whatman CM52 resin (GE Healthcare, Little Chalfont, U.K.): the protein was eluted with the buffer containing 200 mM NaCl. The protein collected was desalted by ultrafiltration using Amicon centricon concentrators (30 kDa cutoff, Merk Millipore Ltd., Cork, Ireland). Reconstitution of BChl a into the B800-Free Protein. BChl a was isolated from the cultured cells of Rhodobacter
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RESULTS Spectral Properties. The B800-free LH2 protein was prepared from a native LH2 protein isolated from Rbl. acidophilus under acidic conditions, which were modified B
DOI: 10.1021/acs.biochem.7b00267 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry from those in previous reports.19,20 In this study, Triton X-100 was used instead of Triton BG-10 as in previous reports,19,20 because Triton BG-10 was not available as an experimental reagent. Triton X-100 plays an active role in the removal of B800 BChl a from the LH2 protein in a buffer containing DDM. BChl a pigments, which were purified from Rba. sphaeroides,26,27 were incubated with B800-free LH2 to produce the reconstituted LH2. Figure 2 shows the electronic
Figure 3. CD spectra of native (black), B800-free (red), and reconstituted LH2 proteins (blue) in the Qy (top) and UV regions (bottom) in 20 mM Tris-HCl buffer containing 0.1% n-dodecyl-β-Dmaltoside.
shaped CD signals between 820 and 900 nm, which originated from the interaction of B850 BChl a pigments, were almost identical in the three proteins. These features of CD signals are also consistent with those reported elsewhere.20 The electronic absorption, fluorescence emission, and CD spectra of B850 BChl a reveal that the removal of B800 BChl a hardly disturbs the orientation and the electronic structures of the πmacrocycles in B850 BChl a. Little change in the CD signals of the three LH2 proteins in the UV region indicates that the contents of the α-helices have similar levels in the three LH2 proteins. Therefore, the local structures in the LH2 protein are barely changed after the removal and insertion of B800 BChl a. Protein Sizes Studied by Size Exclusion Chromatography. The sizes of the B800-free and reconstituted LH2 proteins were analyzed by SEC and compared with those of native LH2 (Figure 4). The native LH2 protein was eluted at 59.0 mL at pH 8.0, and its elution volume was quite similar to that reported under the same analytical conditions.25,31 The B800-free LH2 protein was eluted at 62.6 mL and pH 5.0; the B800-free LH2 eluted later than the native LH2. These results indicate that the overall protein size of the B800-free LH2 becomes small after the removal of B800 BChl a. The elution volumes of the reconstituted LH2 (59.2 mL) were close to those of the native LH2, indicating that the protein size was almost recovered by the insertion of BChl a into the B800 binding sites. The overall sizes of the LH2 proteins were slightly influenced by buffer pH. The elution volume of the native LH2 at pH 5.0 was 60.0 mL, which eluted later than that at pH 8.0. This tendency can be observed in the SEC patterns of both the B800-free and reconstituted LH2. The differences in the elution volumes of the native, B800-free, and reconstituted proteins between pH 8.0 and 5.0 were 1.0, 1.5, and 1.0 mL, respectively. The pH-dependent size changes were further analyzed by the pH titration of simultaneous dynamic light scattering (DLS) and electronic absorption spectroscopy (Figure S2). As a result, the hydrodynamic diameter of the native LH2 slightly decreased around pH 6, although the relative ratio of the absorbance of the B800 band against that of the B850 band was unchanged in this pH region. These results indicate that the slight decrease in the overall size of the LH2 protein under the weakly acidic conditions does not correlate with the removal of
Figure 2. Electronic absorption spectra of native (black), B800-free (red), and reconstituted LH2 proteins (blue) in 20 mM Tris-HCl buffer containing 0.1% n-dodecyl β-D-maltoside. Spectra are offset by 0.5 absorbance unit for the sake of clarity. The inset shows overlapped spectra of native (black) and reconstituted LH2 (blue) in the Qy region.
absorption spectra of the three types of LH2 proteins whose concentrations were adjusted at the Qy peak of the B850 bands. The native LH2 exhibited two intense Qy absorption bands at 802 and 854 nm, which were ascribed to B800 and B850 BChl a, respectively. The B800 Qy band disappeared in the spectrum of the B800-free LH2; the B850 band remained at 855 nm, and its bandwidth was unchanged after the removal of B800 BChl a. The absorbance at the Qy peaks at 802 nm was recovered at 86.7 ± 1.1% of the native LH2 (the average and standard deviation of three samples), which corresponded to the averaged occupancy of 7.8 BChl a pigments in nine B800 binding sites per LH2 protein. This recovery level resembles that reported elsewhere (80%).20 The peak position and bandwidth of the B800 band in the reconstituted LH2 were almost the same as those of the native LH2 (Figure 2, inset). The fluorescence emission properties of B850 BChl a in these LH2 proteins were examined by the excitation of the blueside of the B850 band. The emission peaks of the B850 fluorescence bands were positioned around 870−875 nm in all three proteins (Figure S1). The apparent fluorescence quantum yields of B850 BChl a, which were calculated from the fluorescence bands above 840 nm, were 8.0, 7.4, and 6.0% in the native, B800-free, and reconstituted LH2 proteins, respectively. The slight decrease in the quantum yield induced by the removal of B800 BChl a from LH2 was consistent with a previous report.30 Figure 3 shows the CD spectra of the three types of LH2 proteins in the near-infrared and UV regions. The reversed SC
DOI: 10.1021/acs.biochem.7b00267 Biochemistry XXXX, XXX, XXX−XXX
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B800 BChl a from the protein. The LH2 protein, thus, became compact via a pH decrease regardless of the presence or absence of B800 BChl a, but this change was smaller than that induced by the removal of B800 BChl a. The pH-dependent size decrease, which is not correlated with the removal of B800 BChl a, would originate from the pH-driven local conformational transitions in the regions that have no interactions with BChl a, such as the C-terminus of the α-polypeptides, by the changes of the charges in the amino acid residues.32,33 Other possibilities, including pH-dependent changes in the interactions of the protein surfaces with surrounding detergents, cannot be ruled out. AFM Observations. AFM is a powerful tool for the observation of the morphology of LH2 proteins.34−38 Here we observed three types of LH2 proteins on mica surfaces by a home-built FM-AFM system. The ring structures of all the LH2 proteins are clearly visualized with AFM by the simple physisorption of solubilized LH2 with 0.1% DDM on mica (Figure 5, left). The packing of the B800-free LH2 on mica was different from the packing of the native and reconstituted LH2. These differences would be attributed to the stronger interaction of the solubilized B800-free LH2 with negatively charged mica surfaces at the acidic pH. The circular form and the distribution of the nine units in the three types of LH2 proteins were similar to each other (Figure 5, center). These high-resolution AFM images indicate that no deformation of the LH2 ring structure occurs even if the B800 BChl a pigments are removed from the protein. The height profiles in these AFM observations allow us to compare the ring sizes of
Figure 4. Size exclusion chromatography analysis of native (black), B800-free (red), and reconstituted LH2 proteins (blue) at pH 8.0 (solid curves) and 5.0 (dashed curves). The inset shows overlapped chromatograms of native (black), B800-free (red), and reconstituted LH2 (blue). The proteins were eluted on a HiPrep 16/60 Sephacryl S300 HR column with a mixed buffer of 20 mM Tris and 10 mM succinate containing 0.1% n-dodecyl β-D-maltoside and 150 mM NaCl at a flow rate of 0.4 mL min−1.
Figure 5. High-resolution AFM images of (A) native, (B) B800-free, and (C) reconstituted LH2 proteins adsorbed on mica surfaces. The left column shows wide images. The middle column shows locally enlarged images of single LH2 proteins. Z ranges of the native, B800-free, and reconstituted LH2 are 5, 3, and 2 nm, respectively. The right column shows overlapped height profiles of 10 LH2 proteins. D
DOI: 10.1021/acs.biochem.7b00267 Biochemistry XXXX, XXX, XXX−XXX
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between the elution profiles and the molecular weights of standard samples (Figure S4), are 14 and 18% at pH 8.0 and 5.0, respectively. Note that the AFM analyses presented here do not allow us to discuss the precise vertical shift (several angstrom levels) of the LH2 protein because of the simple preparation of the AFM samples, where the solubilized LH2 proteins were just adsorbed on the mica surfaces. Solubilized LH2 proteins are advantageous for the characterization of the overall protein sizes by the SEC and DLS analyses demonstrated in this study, but further characterization in other systems, such as reconstituted ones in lipid bilayers and detergent/lipid bicelles, is required to obtain more detailed information about LH2 structural changes. AFM measurements of LH2 in membranous systems will particularly allow us to more precisely analyze the structural changes of LH2 by the B800 removal. Specific interactions, which shrink the B800 binding sites after the removal of B800 BChl a, cannot be identified, although the shrinkage of the spaces by van der Waals interactions between amino acid residues and the phytyl chain of B850 BChl a is possible. The insertion of detergents (smaller than the BChl a pigments) into the spaces is another possible mechanism for the decrease in protein size caused by B800 removal. In this study, Triton X-100 was used in the removal of B800 BChl a from LH2, and thus, B800 BChl a might be replaced by this detergent. Pressure-induced spectral changes in the LH2 protein from Rbl. acidophilus and Rba. sphaeroides suggest the presence of two structural domains in the LH2 protein in terms of sensitivity to the applied hydrostatic pressure; one is the hydrophobic domain that includes the B850 binding sites, and the other is the N-terminal domains of both polypeptides in the proximity of the B800 binding sites.11 The domain that includes the B800 binding sites is more sensitive to the applied pressure than those around the B850 binding sites. Therefore, the proximity of the B800 binding sites is flexible, and the local structure is more changeable than the hydrophobic domain around B850 BChl a. In contrast, the hydrophobic domain around B850 BChl a is rather rigid, resulting in little change in the CD signals of B850 BChl a induced by the removal of BChl a. The soft B800 binding pockets will shrink if the BChl a pigments are absent, and the closed form of these pockets mainly originates from the vertical structural changes in the flexible N-terminal domain without deformation of the C9 symmetry circular arrangement of the LH2 protein. Interestingly, the closed B800 binding pockets in the B800-free LH2 protein are smoothly opened by exogenous BChl a pigments and take them up, resulting in a return to a nativelike structure. Therefore, the unique reversibility between the open and closed forms of the B800 binding sites due to the presence and absence of BChl a pigments is the origin of the size changes of the LH2 protein demonstrated in this study. The folding of photosynthetic proteins, especially the mechanism in the organization of pigments with polypeptides, is of great interest in photobiological chemistry, but it has not been thoroughly unraveled yet. Because in vitro assembly of photosynthetic pigments with polypeptides is one powerful methodology for tackling this open question, it has been extensively studied.39−45 In contrast, few studies have addressed the disassembly of pigments from photosynthetic proteins, although such studies can complement the information obtained from the behaviors in the pigment−protein assembly. The effects of the absence of a pigment in photosynthetic
the three types of LH2 proteins. The averaged top-to-top distances of the LH2 ring in the native, B800-free, and reconstituted LH2 were estimated to be 5.2 ± 0.5, 5.4 ± 0.5, and 5.3 ± 0.5 nm (the averages and standard deviations of 10 samples), respectively (Figure 5, right). The ring diameter in the LH2 protein, therefore, did not depend on the presence of the B800 BChl a pigments.
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DISCUSSION We demonstrated the spectral and structural features of the LH2 protein induced by the removal and the insertion of B800 BChl a by spectroscopic, SEC, and AFM analyses. This study indicates the decrease in the overall size of the LH2 protein induced by the removal of the B800 BChl a pigments and the increase in size with the insertion of BChl a into the B800 binding sites with few changes in the local structures around B850 BChl a. The ring structures of B800-free and reconstituted LH2, which were composed of nine units, were visualized for the first time by the AFM measurements presented here. High-resolution AFM images revealed that the LH2 protein kept the nonameric ring structure and its diameter was unchanged even when B800 BChl a was removed from the protein. Previous studies of the reconstitution of B800 BChl a as well as its removal also reported that the absence of B800 BChl a in the LH2 protein did not affect the B850 electronic structure.19−23 This is in line with the results presented here, which indicate that little local structural change occurred in the LH2 protein because of the removal of B800 BChl a. In contrast to no change in the local structure, especially in the B850 region, the overall change in the LH2 structure was induced by the removal of B800 BChl a. This structural change does not originate from the deformation of the nonameric ring structure. Thus, removing B800 BChl a would mainly shrink the B800 binding sites in a direction perpendicular to the πmacrocycle plane of B800 BChl a, resulting in a decrease in the overall size of the LH2 protein demonstrated in this study. We call this decrease in a direction perpendicular to the B800 ring the vertical shift. The vertical shift is hypothesized to be roughly consistent with the distance from the oxygen atom in the axial ligand of the central magnesium in B800 BChl a to the carbon atoms in the phytyl chain of B850 BChl a, which are situated over the central magnesium in B800 BChl a on the other side of the axial ligand (Figure S3), because the distance between the atoms on both sides of the B800 ring is one type of quantitative guidance of plausible shrinkage induced by the removal of B800 BChl a. We estimate this vertical shift from the 3D structure (Protein Data Bank entry 1NKZ). The average distance from the oxygen atom in the axial ligand of the central magnesium in B800 BChl a to four carbon atoms [denoted P9−P12 (Figures S3 and S5)] in the phytyl chain of B850 BChl a is 6.7 Å. The distance of the transmembranous region of the α-polypeptide, which is calculated from the nitrogen atom in αPro12 to the carboxy oxygen atom in αSer36, is 37.2 Å. These two distances concerning the LH2 structure allow us to estimate the level of the vertical shift, induced by the removal of B800 BChl a; it corresponds to 18% of the transmembranous region of the α-polypeptide, which stands almost perpendicular to the membrane. This ratio is in line with the results of SEC analyses. The relative ratios of the decrease in the apparent molecular weights caused by B800 removal to that of the native LH2, which are estimated by the conversion of the SEC elution volumes using the relationship E
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Biochemistry proteins on the protein structures will be helpful in unraveling the structural principle of photosynthetic pigment−protein complexes. From these viewpoints, the structural changes of the LH2 protein induced by the removal and the insertion of B800 BChl a demonstrated in this study will contribute to our understanding of the structural principle and the folding mechanism of photosynthetic pigment−protein complexes. Recently, the structure of the core antenna protein, lightharvesting complex 1 (LH1), derived from a thermophilic photosynthetic bacterium, Thermochromatium tepidum, was reported at 3.0 Å resolution.46 The LH1 protein forms a ring structure of 16 units, each of which consists of α/βpolypeptides with two BChl a pigments and one carotenoid pigment. The bacteriochlorin macrocycles of the two BChl a pigments are stacked in a dimeric form in the LH1 unit, which is analogous to a pair of B850 BChl a pigments in the LH2 protein. The LH1 protein possesses no pigment that corresponds to B800 BChl a. Instead, the LH1 crystal structure exhibited holes on the cytoplasmic side in the transmembranous region of the α/β-heterodimer units. Niwa et al. commented that this region corresponds to the B800 binding site in the LH1 protein.46 The similarity of the positions between the nanospaces in the LH1 and B800 binding sites in the LH2 proteins (Figure S6) in addition to the pigment binding ability of the soft B800 binding pockets in LH2 implies a possibility that B800 BChl a is acquired by such nanospaces in plausible ancestors for integral light-harvesting proteins in the photosynthetic evolution process. Phylogenetic analyses of light-harvesting proteins in purple photosynthetic bacteria suggest that LH2 and LH1 are derived from a common ancestor, although information about the polypeptides in the extant bacterium is somewhat insufficient to conclude this evolutional hypothesis.47−50 Note that the LH2 protein of Phs. molischianum is more closely related to LH1 than the LH2 proteins of other species.48,51 The structural aspects proposed above in addition to phylogenetic analyses will help unravel the evolution of light-harvesting apparatuses. In conclusion, the LH2 protein exhibited reversible changes in the overall size induced by the removal and insertion of B800 BChl a, although no deformation of the C9 symmetry ring structure was induced by the removal of B800 BChl a. The LH2 size changes originate from the shrinkage of the B800 binding sites induced by BChl a removal and the recovery of these nanospaces by the insertion of exogenous BChl a. The remarkable flexibility of the B800 binding sites in the LH2 protein provides useful information for unraveling the mechanisms of the pigment−protein assembly and the photosynthetic functions of this protein as well as the acquisition of B800 BChl a by ancestor proteins in the evolution of photosynthesis. These results will shed new light on the role of chlorophyllous pigments in the architectures of photosynthetic proteins.
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(Figure S3), relationship between Kav values and molecular weights of the standard samples in SEC (Figure S4), molecular structure of BChl a (Figure S5), and overlapped structures of LH2 from Rbl. acidophilus and LH1 from T. tepidum (Figure S6) (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yoshitaka Saga: 0000-0001-7384-9169 Takeshi Fukuma: 0000-0001-8971-6002 Funding
This work was partially supported by JST PRESTO Grant JPMJPR1416 and JSPS KAKENHI Grant JP15H00887 in Scientific Research on Innovative Areas “Artificial Photosynthesis (AnApple)”. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Mr. Ryota Matsui of Kindai University for his experimental assistance. REFERENCES
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00267. Fluorescence emission spectra (Figure S1), pH titration of simultaneous dynamic light scattering measurements and electronic absorption spectroscopy (Figure S2), structure of the B800 binding site in the LH2 protein F
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DOI: 10.1021/acs.biochem.7b00267 Biochemistry XXXX, XXX, XXX−XXX
