Dynamic Interaction between the D1 protein,CP43 and OEC33 at the lumenal side of photosystem II in spinach chloroplasts: evidence from light-induced cross-Linking of the proteins in the donor-side photoinhibition

During the donor-side photoinhibition of spinach photosystem II, the reaction center D1 protein cross-linked with the antenna chlorophyll binding protein CP43 of photosystem II lacking the oxygen-evolving complex (OEC) subunit proteins. The cross-linking did not occur upon illumination of photosystem II samples that retained the OEC33, nor when OEC33-depleted photosystem II samples were reconstituted with the OEC33 prior to illumination. These results suggest that the D1 protein, CP43 and the OEC33 are located in close proximity at the lumenal side of photosystem II, and that the OEC33 suppresses the unnecessary contact between the D1 protein and CP43. Previously we presented data showing the D1 protein located adjacent to CP43 on the stromal side of photosystem II [Ishikawa et al. (1999) BIOCHIM: Biophys. Acta 1413: 147]. The present data suggest that the spatial arrangement of the D1 protein and CP43 at the lumenal side of photosystem II in spinach chloroplasts is similar to that at the stromal side of photosystem II and is consistent with the assignment of these proteins recently proposed on the crystal structures of the photosystem II complexes from cyanobacteria [Zouni et al. (2001) Nature 409: 739, Kamiya and Shen 2003 PROC: Natl. Acad. Sci. USA, 100: 98]. Moreover, the data suggest that the binding condition and positioning of the OEC33 in the photosystem II complex from higher plants may be different from those in cyanobacteria.     

Crystallization of the Photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of <Emphasis Type="Italic">Nicotiana</Emphasis><Emphasis Type="Italic">tabacum</Emphasis>

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b₅₅₉) was purified according to the protocol of Fey et al. (BBA 12:1501–1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group.     

Structural analysis of the photosystem I supercomplex of cyanobacteria induced by iron deficiency

Here we describe the three-dimensional structure of the newly discovered CP43'-photosystem I (PSI) supercomplex of cyanobacteria calculated by single-particle analysis of images obtained by electron cryomicroscopy (cryo-EM). This large membrane protein complex has a molecular mass of approximately 2 MDa and is found in cyanobacteria when grown in iron deficient media. It is composed of a reaction center trimer surrounded by 18 subunits of the chlorophyll a binding CP43'protein, encoded by the isiA gene, which increases the light harvesting capacity of PSI by approximately 70%. By modeling higher-resolution structural data obtained from X-ray crystallography into the three-dimensional (3D) cryo-EM map, we have been able to gain a better understanding of the structure and functional properties of this supermolecular complex. We have identified three separate clusters of chlorophyll molecules at the periphery of the PSI core which may aid energy transfer from the CP43' antenna ring to the reaction center. Moreover, it is shown that despite the replacement of ferredoxin with flavodoxin as an electron acceptor under iron stress conditions, the 3D map has density to accommodate the extrinsic proteins, PsaC, PsaD, and PsaE. The presence of these three proteins was also confirmed by immunoblotting.     

Monovalent cation binding to cubic insulin crystals.

Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution.     

Crystine: fibrous biomolecular material from protein crystals cross-linked in a specific geometry

Cysteine substitutions were engineered on the surface of maltose binding protein to produce crystine fibers, linear polymers of folded protein formed within a crystal. Disulfide bond formation between adjacent protein molecules within the lattice was monitored by X-ray crystallography. The cross-linked crystals were resistant to dissolution in water or neutral buffer solutions, even though the cross-linking was one-dimensional. However, crystine fibers were observed by transmission electron microscopy to dissociate from the crystals in acidic solutions. Some fibers remained associated as two-dimensional bundles or sheets, with a repeat unit along the fibers consistent with the packing of the individual protein molecules in the crystal. Neutralization of the acidic solutions caused the fibers to re-associate as a solid. Crystine threads were drawn out of this solution. In scanning electron microscopy images, many individual fibers could be seen unwinding from the ends of some threads. Crystine fibers are a new type of biomolecular material with potential applications wherever the use of proteins in a fibrous form is desirable, for example, the incorporation of enzymes into cloth or filtration material.     

Revealing the structure of the photosystem II chlorophyll binding proteins, CP43 and CP47

A review of the structural properties of the photosystem II chlorophyll binding proteins, CP47 and CP43, is given and a model of the transmembrane helical domains of CP47 has been constructed. The model is based on (i) the amino acid sequence of the spinach protein, (ii) an 8 A three-dimensional electron density map derived from electron crystallography and (iii) the structural homology which the membrane spanning region of CP47 shares with the six N-terminal transmembrane helices of the PsaA/PsaB proteins of photosystem I. Particular emphasis has been placed on the position of chlorophyll molecules assigned in the 8 A three-dimensional map of CP47 (K.-H. Rhee, E.P. Morris, J. Barber, W. Kühlbrandt, Nature 396 (1998) 283-286) relative to histidine residues located in the transmembrane regions of this protein which are likely to form axial ligands for chlorophyll binding. Of the 14 densities assigned to chlorophyll, the model predicted that five have their magnesium ions within 4 A of the imidazole nitrogens of histidine residues. For the remaining seven histidine residues the densities attributed to chlorophylls were within 4-8 A of the imidazole nitrogens and thus too far apart for direct ligation with the magnesium ion within the tetrapyrrole head group. Improved structural resolution and reconsiderations of the orientation of the porphyrin rings will allow further refinement of the model.     

The 17A structure of the 420 kDa lobster clottable protein by single particle reconstruction from cryoelectron micrographs

Crustaceans form clots by the rapid crosslinking of a hemolymph clottable protein (CP) to form long, branched polymers. Clotting limits hemolymph loss from wounds as well as playing a part in the innate immune response. CP is a 420 kDa homodimer with a large quantity of associated lipid, primarily the carotenoid pigment astaxanthin. The three-dimensional structure of CP from the lobster Panulirus interruptus has been determined to 17 A resolution by single particle reconstruction from electron micrographs of the protein embedded in vitreous ice. The most prominent feature of this structure is a large cavity spanning the length of the molecule, which is the likely lipid binding pocket. The EM structure has been used in a low resolution molecular replacement search with data from orthorhombic CP crystals, and a solution is presented which describes the crystal packing.     

Site-directed mutagenesis of the psbC gene of photosystem II: isolation and functional characterization of CP43-less photosystem II core complexes

Two mutants of Synechocystis PCC 6803 lacking the psbC gene product CP43 were constructed by site-directed mutagenesis. Analysis of cells and thylakoid membranes of these mutants indicates that PS II reaction centers accumulate to a concentration of about 10% of that of WT cells. PS II core complexes isolated from mutants lacking the CP43 subunit show light-driven electron transfer from the secondary electron donor Z to the primary quinone electron acceptor QA with a quantum yield similar to that of wild type, indicating that CP43 is not required for binding or function of QA. The use of mutants for the removal of CP43 thus avoids the loss of QA function associated with biochemical extraction of CP43 from intact core complexes. Both absorbance and fluorescence emission maxima of the mutant complexes show a blue shift in comparison to the WT PS II core complex, indicating that the absorbance spectrum of CP43 is red-shifted relative to that of the remainder of the core complex. The antenna size of these CP43-less complexes is about 70% of that of WT, indicating that approximately 15 chlorophyll molecules are bound by CP43. The molecular mass of the PS II complex, including the detergent shell, shifts from 310 +/- 15 kDa in WT to 285 +/- 15 kDa in the CP43-less mutants.     

抗冻糖蛋白溶液中冰晶生长速率的研究

在分析了溶液中抗冻糖蛋白与冰晶表面的相互作用的基础上,提出了在抗冻糖蛋白溶液中冰晶沿c轴方向生长的理论。给出了冰晶在抗冻糖蛋白溶液中生长速率的定量计算,而且理论值与实验结果有较好的符合,解释了冰晶在抗冻糖蛋白溶液中生长速度和生长习性的各向异性。     

Nomenclature for membrane-bound light-harvesting complexes of cyanobacteria

Accessory chlorophyll-binding proteins (CBP) in cyanobacteria have six transmembrane helices and about 11 conserved His residues that might participate in chlorophyll binding. In various species of cyanobacteria, the CBP proteins bind different types of chlorophylls, including chlorophylls a, b, d and divinyl-chlorophyll a, b. The CBP proteins do not belong to the light-harvesting complexes (LHC) superfamily of plant and algae. The proposed new name of CBP for this class of proteins, which is a unique accessory light-harvesting superfamily in cyanobacteria, clarifies the confusion of names of prochlorophytes chlorophyll binding protein (Pcb), PSII-like light-harvesting proteins and iron-stress-induced protein A (IsiA). The CBP complexes are a member of a larger family that includes the chlorophyll a-binding proteins CP43 and CP47 that function as core antennas of photosystem II.     

The role of CP 47 in the evolution of oxygen and the binding of the extrinsic 33-kDa protein to the core complex of Photosystem II as determined by limited proteolysis

In order to identify the domain within Photosystem II complexes that functions in the evolution of oxygen, we performed limited proteolysis with lysylendopeptidase of the core complex of Photosystem II which had been depleted of the extrinsic 33-kDa protein (Mn-stabilizing protein). The cleavage sites were estimated from the amino-terminal sequences of the degradation fragments, their apparent molecular masses and amino-acid compositions. Under certain conditions, the D2 protein was cleaved at Lys13; and a chlorophyll a-binding protein, CP 47, was cleaved at Lys227 and Lys389. Another chlorophyll a-binding protein, CP 43, was degraded more rapidly than CP 47. The oxygen-evolving activity and the capacity for rebinding of the 33-kDa protein to the core complex of Photosystem II decreased in parallel, with kinetics very similar to those of the cleavage of CP 47 at Lys389. These observations strongly suggest that the hydrophilic domain around Lys389 of CP 47, which are located on the lumenal side, is important in the binding of the 33-kDa protein and in maintaining the oxygen-evolving activity of the Photosystem II complex.Abbreviations CP 47 and CP 43- intrinsic chlorophyll a-binding proteins with apparent molecular masses of 47 and 43 kDa, respectively - PBQ- phenyl-p-benzoquinone - TLCK- N--p-tosyl-L-lysine chloromethyl ketone     

Three-dimensional model and characterization of the iron stress-induced CP43'-photosystem I supercomplex isolated from the cyanobacterium Synechocystis PCC 6803

The cyanobacterium Synechocystis PCC 6803 has been subjected to growth under iron-deficient conditions. As a consequence, the isiA gene is expressed, and its product, the chlorophyll a-binding protein CP43', accumulates in the cell. Recently, we have shown for the first time that 18 copies of this photosystem II (PSII)-like chlorophyll a-binding protein forms a ring around the trimeric photosystem I (PSI) reaction center (Bibby, T. S., Nield, J., and Barber, J. (2001) Nature, 412, 743-745). Here we further characterize the biochemical and structural properties of this novel CP43'-PSI supercomplex confirming that it is a functional unit of approximately 1900 kDa where the antenna size of PSI is increased by 70% or more. Using electron microscopy and single particle analysis, we have constructed a preliminary three-dimensional model of the CP43'-PSI supercomplex and used it as a framework to incorporate higher resolution structures of PSI and CP43 recently derived from x-ray crystallography. Not only does this work emphasize the flexibility of cyanobacterial light-harvesting systems in response to the lowering of phycobilisome and PSI levels under iron-deficient conditions, but it also has implications for understanding the organization of the related chlorophyll a/b-binding Pcb proteins of oxychlorobacteria, formerly known as prochlorophytes.     

Structure of light-harvesting chlorophyll a/b protein complex from plant photosynthetic membranes at 7 A resolution in projection

The structure of thin three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex, an integral membrane protein from the photosynthetic membrane of chloroplasts, has been determined at 7 A (1 A = 0.1 nm) resolution in projection. The structure analysis was carried out by image processing of low-dose electron micrographs, and electron diffraction of thin three-dimensional crystals preserved in tannin. The three-dimensional crystals appeared to be stacks of two-dimensional crystals having p321 symmetry. Results of the image analysis indicated that the crystals were disordered, due to random translational displacement of stacked layers. This was established by a translation search routine that used the low-resolution projection of a single layer as a reference. The reference map was derived from the symmetrized average of two images that showed features consistent with the projected structure of negatively stained two-dimensional crystals. The phase shift resulting from the displacement of each layer was corrected. Phase shifts were then refined by minimizing the phase residual, bringing all layers to the same phase origin. Refined phases from different images were in agreement and reliable to 7 A resolution. A projection map was generated from the averaged phases and electron diffraction amplitudes. The map showed that the complex was a trimer composed of three protein monomers related by 3-fold symmetry. The projected density within the protein monomer suggested membrane-spanning alpha-helices roughly perpendicular to the crystal plane. The density in the centre and on the periphery of the trimeric complex was lower than that of the protein, indicating that this region contained low-density matter, such as lipids and antenna chlorophylls.     

Changes of absorption spectra during heat-induced denaturation of Photosystem II core antenna complexes CP43 and CP47: revealing the binding states of chlorophyll molecules in these two complexes

The Photosystem II (PSII) core antenna complexes, CP43 and CP47, were prepared from spinach (Spinacia oleracea L.). The absorption spectra in the red region at room temperature were recorded for the PSII core antenna samples after increased temperature treatment (up to 80 degrees C). Derivative and difference spectra revealed the existence of two groups of chlorophyll a (Chl a) molecules in both CP43 and CP47. The one with the absorption peak in the shorter wavelength region was designated as CP43-669 and CP47-669, while the other with the absorption peak in the longer wavelength region was designated as CP43-682 and CP47-680. The results of the thermal treatment experiment demonstrated that CP43-669 and CP47-669 may exist as monomers of Chl a and that their binding sites on the polypeptides are insensitive to thermal treatment, whereas CP43-682 and CP47-680 may exist as dimers or multimers of Chl a and their binding regions in the polypeptide chains are more sensitive to heat treatment. The excitation energy transfer mechanism between these two different groups of Chl a molecules is also analyzed.     

Control of crystal forms of apoferritin by site-directed mutagenesis

Surface charges of protein molecules are not only important to biological functions but also crucial to the molecular assembly responsible for crystallization. Appropriate alteration in the surface charge distribution of a protein molecule induces new molecular alignment in the proper direction in the crystal and, hence, controls the crystal form. Apoferritin molecules are known to crystallize in two- and three-dimensional forms in the presence of cadmium ions, which bridge neighboring protein molecules. Here we report a controlled transformation of the apoferritin 2-D crystal by site-directed mutagenesis. In mutant apoferritin, two amino acid residues binding a cadmium-ion through their negative charge, were replaced by one type of nonionic amino acid residues. The amino acid residues, Asp-84 and Gln-86 in the sequence of recombinant (i.e., wild-type) horse L -apoferritin, were replaced by Ser. The wild-type apoferritin yielded a hexagonal lattice 2-D crystal in the presence of cadmium ions. In contrast, the mutant apoferritin yielded two types of oblique crystals independent of the presence of cadmium ions. Image reconstruction of electron micrographs of the mutant crystals made clear that the mutant apoferritin molecules oriented themselves with the 2-fold symmetry axis perpendicular to the crystal plane in both crystals, while the wild-type apoferritin molecules oriented themselves with the 3-fold symmetry axis perpendicular to the crystal plane. The changes of crystal forms and molecular orientation in the 2-D crystals were well explained by a change of the electrostatic interactions induced by the mutagenesis. © 1995 Wiley-Liss, Inc.     

Molecular topology of the Photosystem II chlorophyll a binding protein,CP 43: Topology of a thylakoid membrane protein

We have used antibodies generated against synthetic peptides to determine the topology of the 43 kD chlorophyll a binding protein (CP 43) of Photosystem II. Based on the pattern of proteolytic fragments detected (on western blots) by peptide specific antibodies, a six transmembrane span topological model, with the amino and carboxyl termini located on the stromal membrane surface, is predicted. This structure is similar to that predicted for CP 47, a PS II chlorophyll a binding protein (Bricker T (1990) Photosynth Res 24: 1–13). The model is discussed in reference to the possible location of chlorophyll binding sites.This work was supported by National Institutes of Health Research Grant, GM40703 and U.S. Department of Energy Grant, DE-FG01-92ER20076 (to R.T.S.).     

Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B₁₂. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.     

Ligand-linked structural transitions in crystals of a cooperative dimeric hemoglobin

Cooperative ligand binding in the dimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis is mediated primarily by tertiary structural changes, but with a small quaternary rearrangement (approximately 3 degrees), based on analysis of distinct crystal forms for ligated and unligated molecules. We report here ligand transition structures in both crystal forms. Binding CO to unligated HbI crystals results in a structure that approaches, but does not attain, the full allosteric transition. In contrast, removing CO from the HbI-CO crystals results in a structure that possesses all the key low affinity attributes previously identified from analysis of HbI crystals grown in the unligated state. Subsequent binding of CO shows the reversibility of this process. The observed structural changes include the quaternary rearrangement even under the constraints of lattice interactions, demonstrating that subunit rotation is an integral component of the ligand-linked structural transition in HbI. Analysis of both crystal forms, along with data from HbI mutants, suggests that the quaternary structural change is linked to the movement of the heme group, supporting a hypothesis that the heme movement is the central event that triggers cooperative ligand binding in this hemoglobin dimer. These results show both the effects of a crystal lattice in limiting quaternary structural transitions and provide the first example of complete allosteric transitions within another crystal lattice.     

Crystals of Lumbricus erythrocruorin

Lumbricus terrestris erythrocruorin, a 3.9 X 10(6) Mr respiratory protein, has been crystallized in four different forms. Despite the high molecular symmetry apparent from images in electron micrographs, only one crystal form expresses any molecular symmetry as crystallographic symmetry. The lattice parameters provide upper limits on the molecular dimensions of 267 A X 308 A X 172 A (1 A = 0.1 nm), which agree well with dimensions obtained from electron micrographs of negatively stained molecules. We have collected diffraction data to 5.5 A from type III crystals and have begun a structural analysis.     

Enzymatic function in crystals of delta 5-3-ketosteroid isomerase. Catalytic activity and binding of competitive inhibitors

Crystals of the steroid-metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni, exhibit many enzymatic properties. Each enzyme subunit in the lattice binds a competitive inhibitor, progesterone, with the same stoichiometry (1:1) and affinity (KD = 6 X 10(-6) M) as the enzyme in solution. Another competitive inhibitor, 19-nortestosterone, competes with progesterone for the same binding sites in the crystal. The enzyme crystals catalyze the conversion of delta 5- to delta 4-ketosteroids, but because the enzyme is so efficient, and substrate diffusion into the crystal is so slow, substrate cannot penetrate deeply into the crystal before being converted to product. A general theoretical formulation is presented to account for the effects of substrate diffusion into enzyme crystals of different shapes and sizes. The dependence of apparent mean enzyme activity in steroid isomerase crystals as a function of crystal size is shown to be consistent with this theoretical formulation. These inhibitor binding and catalytic properties suggest that the enzyme is in an active conformation within these crystals.