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Biological Research
versão impressa ISSN 0716-9760
Biol. Res. v.37 n.4 supl.A Santiago 2004
doi: 10.4067/S0716-97602004000500003
| Biol Res 37: 733-745, 2004 ARTICLES Structural-functional analysis of the oligomeric protein R-phycoerythrin
JOSÉ MARTÍNEZ-OYANEDEL1,3, CARLOS CONTRERAS-MARTEL2, CAROLA BRUNA1 and MARTA BUNSTER1
1 Laboratorio de Biofísica Molecular, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Chile Dirección para Correspondencia ABSTRACT
The structure of phycobiliproteins and their spatial organization in the phycobilisome provide the environment for high efficiency in light harvesting and conduction towards photosystem II. This article focuses on the analysis of R-phycoerythrin, a light harvesting hexameric phycobiliprotein that is part of the phycobilisomes. The interaction surfaces and the environment of the chromophores of R-phycoerythrin were studied in order to explain its structural stability and spectroscopic sensitivity, properties revealed by perturbation experiments. Three interaction surfaces are described (ab), (ab)3 and (ab)6. The analysis shows the importance of a subunits in the interaction between trimers, the homodimeric nature of the monomer (ab) and also the presence of anchor points in every interaction surface studied: a18Phe and b18Tyr for (ab), b76Asn for (ab)3 and a25Asn for (ab)6 . Side chains of arginine, lysine or glutamine residues are located in the proximity of the chromophores providing the correct stabilization of their carboxylates. Aspartic acids residues are associated through H-bonds to the N atom of the two central rings of the tetrapyrrolic chromophores. Changes in the spectroscopic properties are observed in perturbation experiments, confirming the spatial requirement for an efficient resonance energy transfer among chromophores and through the phycobilisome.
Key terms: 1eyx, anchor points, interaction surfaces, stability, spectroscopic sensitivity INTRODUCTION
A process of resonance energy transfer with the highest efficiency reported in biological systems occurs in phycobilisomes (PBS) present in red alga and cyanobacteria (21). This macromolecular complex is capable of harvesting and conducting light with an efficiency over 95% in the energy transfer process (13). The protein components of PBS, the phycobiliproteins (PBPs), belong to a family of chromophorylated highly-fluorescent proteins (13) that provide a protein network to which the chromophoric groups are covalently bound for light conduction. Phycobiliproteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC) are structurally very similar; their principal difference consists in the quality and quantity of their chromophoric groups. PBPs are arranged as a heterodimer (ab) that oligomerizes to (ab)3 in APC and to (ab)6 for PE and PC (13). A scheme for these different oligomerization states is shown on Figure 1A. The phycobilisome is composed of a central core of APC, which is associated with photosynthetic membranes and peripheral rods that contain PC proximal to the core and PE in the distal part of the rod (11). They are assembled by linker polypeptides, which provide the structural environment for its function and stabilization (8, 10). Each PBP performs a specific role in the transfer chain reflected by their position in the rods and by the chromophores they contain. The high efficiency in the energy transfer depends on the correct packing of the oligomers, i.e. the functional unit of each PBP and the precise organization in the PBS. Recently, we solved the crystal structure of R-Phycoerythrin (R-PE) (PDB Code: 1eyx) from Gracilaria chilensis at 2.2Å resolution (8). R-PE maintains the structural features observed in other phycobiliproteins: a globin-like folding, the association of a and b subunits, and the position of the cysteines that bind the chromophores, as shown in Figure 1B (1, 3, 6, 7, 9, 14, 21). In this article, we performed a structural analysis of R-PE as a model for studying the importance of protein-protein interactions and the properties of the surfaces and the residues involved in the conservation of the geometry of the chromophores. This analysis will allow us to understand the high stability of this protein and the great sensitivity of its spectroscopic properties to environmental conditions (5, 7), providing important aspects to consider when mimicking biological systems for biotechnological purposes.
MATERIAL AND METHODS A) Materials
Gracilaria chilensis was collected from the Pacific Coast at the Marine Biology Station of the Universidad de Concepción in Dichato (36º32' S, 72º57' W). The reagents used were pro-analysis (MERCK, Darmstadt, Germany).
B) Methods
1. Purification and characterization: R-phycoerythrin for the perturbation experiments was purified according to literature (4). The purity of the protein was analyzed by SDS-PAGE 10% (17) with a 6% stacking gel and visualized using Coomassie Blue R-250 stain (20). Dilutions of this stock protein (3mg/ml) were used in all the experiments. 2. Fluorescence and absorption spectroscopy: The differential absorption spectroscopy was performed in a Shimadzu UV-160 spectrophotometer. Ultraviolet and visible spectra were recorded. Samples (dilution 1:20) were incubated for 10 min at the different temperatures in phosphate buffer 100mM pH 6.0 or in acetate buffer 100mM pH 4.0. Fluorescence spectroscopy was performed using a Shimadzu fluorimeter RF-5301 PC with dilution 1:250. The emission was recorded after the incubation of the samples for 10 min at the same conditions of temperature and pH. The excitation wavelengths were 566nm, 550nm and 495nm, the three maxima of the R-phycoerythrin spectrum (4). Slits of 1.5nm for absorption and emission spectra were used.
C) Structural analysis: The surface accessible area (12) was calculated from the coordinates of R-PE of Gracilaria chilensis (PDB code: 1eyx), using a 1.4 Å probe. Hydrogen bond distances and contact distances were obtained using HBPLUS v.3.51 (18). The electrostatic surface representations were generated using WebLab ViewerPro v3.5 (Molecular Simulation Inc.). RESULTS
A) Description and analysis of the interaction surfaces established by the three-dimensional packing of R-PE
1) Monomer Surface ab: This surface is formed by helices X, Y and A, loop X-Y and part of helix E of the a subunit, and by N-terminal residues of helices X, Y and A, loop X-Y, part of helices E and F, and the chromophore b158PEB of the b subunit (Figure 1B). The assignation of the helical segments is according to literature (24). A very poor distribution of charges is observed (Figure 2A and 2B), and they are located in the outer surface of the subunits. It is also a highly apolar interface (54% and 60% of the a and b subunits interface are hydrophobic residues, respectively), composing an hydrophobic core. The few polar residues involved in the interaction surface are a3Ser, a13Asp, a42Glu, a91Arg, b3Asp, b13Asp and b91Arg. All of them are highly conserved in evolution (2). The buried residues are located on helices X and Y, where the lateral chains are interlocked. a18Phe and b18Tyr also are clearly defined as anchor points for the interaction surface, as shown in Figure 3A.
The total buried surface upon package of the monomer ab is 3003Å2 (14.9%). This interaction surface shows a homodimeric nature (binary axis), which was expected from the sequence and structural homology between both subunits.
2) Trimer surface (ab)3: This surface is formed by a repetition of ab around a threefold axis and produces two new interaction surfaces: - 1a-3b is an ab type interface and is formed by helices E, F and F', loop E-F and the chromophore a82PEB from the 1a subunit (see Figure 1B for nomenclature) and helices B and E, loop B-E and the chromophore b50/51PUB from the 3b subunit. The interaction surface in the a subunit shows an uncharged core surrounded by a group of charged residues (a81Lys, a84Arg and a88His) in the vicinity of aPEB82, which is also near bPUB50/61 of the next monomer (Figure 2C). The b subunit also contributes to this surface with a narrow stretch of polar residues such as b76Asn, which is deeply buried in the a subunit acting as an anchor point (Figure 3B), and b77Arg located towards a107Glu, a111Ala and a112Gly at H-bond distance to a82PEB (Figure 2D). - 1b-2b is a bb type interface, where residues from helix X and loop X-Y from the 1b subunit interact with helix E and loop B-E of the 2b subunit. The interaction surface is quite minimal and it is stabilized by an H-bond between b74Tyr and b13Asp. The total accessible surface internalized upon trimer oligomerization is 3218 A2 (6.1%). 3) Hexamer surface (ab)6: The hexamer can be described as two trimers related by a binary axis. The interaction surfaces are mainly established through a subunits with only a minor contribution of b subunits (Figure 2E, 2F), describing three components: - 1a-6a, composed by residues from helices X, Y and H, loops X-Y and E-F from both chains. Helices X and Y are orthogonally related to their homologs in the other a subunit. Uncharged residues are buried and surrounded by charged residues. The residue 1a25Glu penetrates deeply in 6a, interacting with 6a37Arg (Figure 3C). As mentioned earlier, all the surfaces present anchor points formed by particular residues, deeply buried in the opposite face. - 1a-5a involves helices F' and G, loops B-E and F`-G of both chains. The residue 1a71Glu located between 5a62Lys and 5a63Tyr forms an H-bond with 5a63Tyr. 1a65Tyr interacts with its analogue residue 5a65Tyr. In addition, 1a114Arg is parallel to 5a114Arg and 5a118Arg (Figure 2C) and forms a H-bond with 5a115E. The charged residues a62Lys, a71Glu, a114Arg, a115Glu and a118Arg distribute as a triangle. Its center presents a small area of uncharged residues forming another hydrophobic patch. - 1a-6b involves helix H, loops B-E and G-H and aPEB139 of the a subunit and residues from the b subunit belonging to helix A, loop A-B and bPEB158. In this surface b49Ser and b46Ser are interlocked with a164Ser and a161Asn. b149Arg is located between a157Asp and a135Arg, producing a polar region. bPEB158 is stabilized by H-bonds with a147Gln and a33Arg. It is not possible to describe any hydrophobic patch on this surface. The total accessible surface internalized upon hexamer oligomerization is 11968Å (12.4%).
B) Description and analysis of the hydrogen bonds formed by the hexamer oligomerization
In R-PE, 162 intrachain H-bonds were detected for the a subunit and 172 for the b subunit (Table I). The a/b interaction surface presents 14 H-bonds, in which the H-bond between a28Gln and bPEB158 is the most important. In the (ab)3 interaction surfaces, only three H-bonds were detected between each monomer. Fifteen interchain H-bonds were observed in the dimer (ab)2, most of them involving a chains.
C) The chromophores
The spectroscopic properties of phycoerythrobilin (PEB) and phycourobilin (PUB), the chromophores present in R-PE, depend on their chemical constitution and environment (7). Table II shows the environment of the chromophores in R-PE. It is possible to appreciate that 1aPEB82 (Chain A) is located towards the inner part of the ring (see Figure 1A and reference 8). This chromophore makes van der Waals contacts with six residues from different chains and participates in one interchain H-bond. 1bPEB82 (Chain B) is also located towards the center of the ring and is exposed to solvent. It interacts with 1aPEB82 and the modified residue N-methyl asparagine 72. aPEB139 is located at the periphery of the hexamer and establishes three H-bonds. bPEB158 is also located towards the periphery of the hexamer, strongly H-bonded as described earlier (Table II). Besides residues from the b chain, two amino acids from an a chain of the other half of the dimer participate in the stabilization of bPEB158, which explains the deep changes in the spectroscopic properties produced by small structural changes. b50/61PUB is buried in the protein and do not form any interchain H-bond.
Table II, Cont.
All the chromophores are hydrogen bonded to aspartic groups by the atoms NA and ND of the central rings, as shown in Table I. a82PEB and b50/61PUB are in proximity to one arginine; a139PEB is close to two Arg and b82PEB is close to three arginine residues.
D) Perturbation experiments
Table III shows that when the temperature was increased from 30 to 70ºC, a decrease of the fluorescence intensity was observed. There is also a decrease in the lmax of absorbance of PUB (495nm) that persists until 40ºC, remaining constant at higher temperatures at both pH studied, as shown in Figure 4.
DISCUSSION
The main forces for the packing of oligomeric proteins are hydrophobic interactions, surface complementarity, van der Waals interactions and electrostatic interactions, such as H-bonds and salt bridges (19, 29). Hydrogen bonds participate in the stabilization of quaternary structures. Their perturbation would produce alterations in any protein-protein interaction (23, 30). In a particular geometry, donor and acceptor can establish an H-bond interaction, which are highly specific and show additive and cooperative forces (16). This is the case of R-PE. The interaction between a and b subunits is extremely strong and requires pH 2 and 9M urea for dissociation (26). It involves wide hydrophobic patches and 14 H-bonds. The ab interaction surface resembles the interaction of homodimer surfaces (15) and also behaves as such. This fact would explain the extreme pH and concentrations of urea necessary to separate the a and b subunits. The association of monomers into trimers involves only 9 H-bonds and some hydrophobic patches. The packing of the hexamer by the association of two trimers involves hydrophobic patches and 45 H-bonds. These structural characteristics agree with evidence reported previously (5) in which increasing concentrations of urea from 0 to 6M produce a serial decrease of the absorbance at 550 and 566nm (phycoerythrobilin chromophores), indicating the destabilization of the monomers (26). A minor change was observed at 540nm (lmax PUB). Changes in the pH, from 6.0 to 4.0, should theoretically cause the protonation of the aspartic acids, residues that are related to the stabilization of the conformation of each chromophore. This change in conformation could be responsible for a decrease in the efficiency of the resonance energy transfer, which is reflected on the spectroscopic properties of the protein. This idea agrees with the results of the experiments performed to test the stability of R-PE. A decrease in pH from 6.0 to 4.0 may also interrupt some of the H-bonds crucial for the stability of the structure of the hexamer. Apparently, the dissociation of the hexamer in trimers is induced at pH 4.0. This could be explained by the involvement of aspartic or glutamic acids in 24 of the 45 H-bond distances. These residues would be close to their dissociation equilibrium, producing a weak association by the decrease in the number of contacts between trimers (5). The effect of increasing temperatures or urea concentrations on the dissociation of the hexamer, can be attributed mainly to the weakening of the aa interactions. Hydrophobic interactions also are affected strongly by changes of the solvent polarity, as well as temperature or urea concentration increments (5, 29). Despite the extremely high structural stability of its oligomerization state (ab)6, R-PE presents spectroscopic sensitivity, which is evident from the quenching of the fluorescence observed when the three-dimensional structure is altered and local conformational modifications are produced which affect its spectroscopic properties. In this sense, two important aspects must be considered: the effect of perturbations in the amino acid residues interacting with the chromophores and the importance of the oligomerization state for the conformation of the chromophores. The carboxylate groups of the protein involved in the stabilization of the conformation of the chromophores should be affected by changes in pH. Experimental data confirm this fact, as shown in Figure 4. At pH 6.0 and pH 8.0 (data not shown), the protein is stable and maintains its spectroscopic properties. Whereas at pH 4.0, where the carboxylates of aspartic or glutamic acids are close to the dissociation equilibrium, a very fast decrease of the absorbance and fluorescence occurs. This may be related to the perturbation of the H-bonds formed by these residues with the chromophores, producing a change in their conformation and inhibiting the resonance energy transfer through the molecule. An additional effect could be produced by the carboxylates present in the chromophores, which would affect their dipolar moment and hence, the efficiency of the energy transfer. pH perturbation was shown to affect the stability of the interaction between trimers, inducing the dissociation of the hexamer (5). Chromophore 1b158PEB (chain B) is stabilized by H-bonds by 6a33Arg and 6a147Gln of chain K. Thus, its spectroscopic properties should be affected by dissociation. Changes in pH should also produce the protonation of the residues a3Asp and a13Asp involved in the ab interaction and b13Asp involved in the interaction ab/ab in (ab)3. This change of pH would also affect residues a25Glu, a106Asp, a157Asp, a71Glu, and a115Glu, which are important for the stabilization of the hexamer. The changes in absorbance observed consist of a fast decrease of the absorbance at 566nm and 545nm (PEB) with a slower decrease on the maximum at 495nm (PUB). PUB is bound through rings C and D to bCys50 and bCys61, while PEB is bound to the protein only through one cysteine, explaining its higher sensitivity. The quenching of the fluorescence could be produced by a combination of local and global perturbations. Changes in conformation and in the conjugation degree of the chromophore produced by acid pH may expose the chromophores to solvent producing the quenching of the fluorescence. This effect is accelerated by an increase of the temperature (Table III).The comparative analysis of the conformation and environment of the chromophores of R-phycoerythrin from G. chilensis (8) (1eyx.pdb) at 2.2Å resolution and R-PE from Polysiphonia. urceolata at 2.8Å resolution (6) (1lia.pdb) and from Griffitsia molinis at 1.9Å resolution (22) (1b8d.pdb) indicate that chromophores 1a82PEB, 1b82PEB and 1b50/61PUB present very similar conformation and environment. On the other hand, chromophore 1a139PEB shows less similarity among these three proteins probably due to the flexibility of the loop 139-146, which is bound to the chromophore and provides an important part of the contact surface. Chromophore 1b158PEB presents a higher conformational difference, which could be induced by the change in conformational conformers of V153 and I154. The most important differences were observed for the carboxylic groups of each chromophore. Nevertheless, the conjugation occurs only through the tetrapyrrol part of the chromophores structure. Therefore, this difference should not have major influence on the process of resonance energy transfer. This structural analysis revealed the following structural requirements for a correct resonance energy transfer common to PE from other species and also to other PBPs (2, 25): 1. Side chains of arginine, lysine or glutamine residues located in the proximity of the chromophores, to provide the correct stabilization of the carboxylates through their ionizable groups. 2. Aspartic acids associated to the central rings of the chromophore by three centered H-bonds. The rest of the chromophore interactions are mainly van der Waals contacts, so their conformation will be affected by changes of polarity or solvent hydrophobicity. 3. Chromophores in a restricted conformation necessary to perform the function of conducting light through R-PE and direct it through the rest of the antenna. The presence of phycobilins associated to the apoprotein also has been reported to be important for the correct packing of phycobiliproteins (28). 4. High complementarity between subunits, as shown in Figure 2, with presence of anchor points in every interaction surface (some of them shown in Figure 3), which contribute to the hexamer structural stability. More structural information is required to confirm if the g subunit also is contributing to the stabilization of the chromophores. Nevertheless, the structure reported for allophycocyanin complexed with a linker polypeptide (21) shows that the linker provides a hydrophobic environment to the chromophore exposed to solvent in the inner part of the ring. Some of these residues are in a similar position with the available residues of the g subunit (Chain G) in the structure of phycoerythrin (8). Hence, the g subunit should have a similar role in the complex. This analysis has provided interesting information about which aspects need to be considered to preserve the geometry of the phycobilins and the protein network to fulfill the requirements for a perfect resonance energy transfer in this system. Theoretical studies on the effect of geometry of the chromophore and their environment in the transfer constant are in progress.
ACKNOWLEDGMENTS
This work was supported by funding from the Dirección de Investigación de la Universidad de Concepción, Grants Nº 99.031.084-1.0 and 202.031.091-1.0.
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Received: February 27, 2004-10-22. In Revised Form: July 27, 2004-10-22. Accepted: September 2, 2004 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
