The structure and function of CP47 and CP43 in Photosystem II

This Minireview presents a summary of recent investigations examining the structure and functions of the Photosystem II chlorophyll-proteins CP47 and CP43, updating our previous review which appeared in 1990 (TM Bricker, Photosynth Res 24: 1–13). Since this time, numerous studies have clarified the roles of these chlorophyll-proteins within the photosystem. Biochemical, molecular and structural studies (electron and X-ray diffraction) have demonstrated the close association of these components with the photochemical reaction center of the photosystem and with the extrinsic oxygen evolution enhancer proteins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modeling and simulation of the initial phases of chlorophyll fluorescence from Photosystem II

A simple kinetic model structure for chlorophyll fluorescence (ChlF) from Photosystem II (PSII) offers practical usefulness in quantitative analysis and extraction of information from measured ChlF. In this work, the major PSII phototransduction kinetics was represented with only five state variables. Parameters were estimated through a least-squares algorithm. The developed model structure could produce the well-known OJIP pattern and fit measured ChlF. Influences of PQ pool size, active Q_B sites, and Q_A reduction rate on ChlF emission were simulated and discussed in light of the existing literature.     

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.     

The role of axial ligands for the structure and function of chlorophylls

We have studied the effect of axial ligation of chlorophyll and bacteriochlorophyll using density functional calculations. Eleven different axial ligands have been considered, including models of histidine, aspartate/glutamate, asparagine/glutamine, serine, tyrosine, methionine, water, the protein backbone, and phosphate. The native chlorophylls, as well as their cation and anion radical states and models of the reaction centres P680 and P700, have been studied and we have compared the geometries, binding energies, reduction potentials, and absorption spectra. Our results clearly show that the chlorophylls strongly prefer to be five-coordinate, in accordance with available crystal structures. The axial ligands decrease the reduction potentials, so they cannot explain the high potential of P680. They also redshift the Q band, but not enough to explain the occurrence of red chlorophylls. However, there is some relation between the axial ligands and their location in the various photosynthetic proteins. In particular, the intrinsic reduction potential of the second molecule in the electron transfer path is always lower than that of the third one, a feature that may prevent back-transfer of the electron.     

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.).     

The insecticidal crystal protein Cry2Ab10 from Bacillus thuringiensis: cloning, expression, and structure simulation

The cry2Ab-type gene was cloned from Bacillus thuringiensis and designated as cry2Ab10. The recombinant Cry2Ab10 protein expressed in E. coli cells shows high toxicity against Plutella xylostella. The protein structure was constructed by homology modeling, and the receptor-binding sites were predicted by a molecular docking method.     

Genetic analysis of the Photosystem I subunits from the red alga, Galdieria sulphuraria

Currently, there are very little data available regarding the photosynthetic apparatus of red algae. We have analyzed the genes for Photosystem I in the recently sequenced genome of the red alga Galdieria sulphuraria. All subunits that are conserved between plants and cyanobacteria were unambiguously identified in the Galdieria genome: PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK and PsaL. From the plant specific subunits, PsaN and PsaO were identified but the sequence homology was much lower than for the subunits that are present in plants and cyanobacteria. The subunit PsaX, which is specific for thermophilic cyanobacteria, is not present in the Galdieria genome, whereas PsaM is a plastid-encoded protein as in other red algae. The sequences of the core subunits of PSI were further analyzed by mapping of the conserved areas in the crystal structures of cyanobacterial and plant PSI. The structural comparison shows that PSI from the red alga Galdieria may represent a common ancestral structure at the interface between cyanobacterial and plant PSI. Some subunits have a “zwitter” structure that contains structural elements that show similarities with either plant or cyanobacterial PSI. The structure of PsaL, which is responsible for the trimerization of PSI in cyanobacteria, lacks a short helix and the Ca²⁺ binding site, which are essential for trimer formation indicating that the Galdieria PSI is a monomer. However the sequence homology to plant PsaL is low and lacks strong conservation of the interaction sites with PsaH. Furthermore, the sites for interaction of plant PSI with the LHCI complex are not well conserved between plants and Galdieria, which may indicate that Galdieria may contain a PSI that is evolutionarily much more ancient than PSI from green algae, plants and the current cyanobacteria.     

Isolation and crystallization of CP47, a Photosystem II chlorophyll binding protein. Degradation of CP47 upon dissociation from the core complex

The CP47 protein was isolated from Photosystem II membranes by using a combination of the detergents n-dodecyl-β-D-maltoside and octyl-β-D-thioglucoside. The purified CP47 was used in a series of crystallization experiments, which yielded highly reproducible hexagonal crystals. Immunoblot analysis revealed that the isolated CP47 undergoes degradation even under dim light conditions. This degradation takes place after the protein has been dissociated from the core complex. Proteolysis experiments with trypsin demonstrated that the dissociation of the CP47 from the PS II core complex results in changes that render the protein sensitive to proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Design,synthesis, crystal structure,biological evaluation and molecular docking studies of carbazole-arylpiperazine derivatives

Subtype-selective α₁-adrenoceptor (AR) antagonists display optimum therapeutic efficacies for the treatment of benign prostatic hyperplasia (BPH). In this study, we designed and synthesized novel carbazole-arylpiperazines derivatives (1 and 2) on the basis of the proposed pharmacophore model for α₁-AR antagonists. Structural properties were investigated using single-crystal X-ray diffraction analysis. Comparison of crystal structures with ligand-based pharmacophore models revealed that the two agents may possess antagonistic effects on α_1D subtype. Tissue functional assay in vitro showed that compound 2 exerted strong antagonistic activity on α_1B-AR (pA₂ 7.13) with a poor selectivity for α_1A and α_1D subtypes. Compound 1 exhibited enhanced antagonistic effect on α_1D subtype (pA₂ 7.06) and excellent selectivity for α_1D over α_1B (α_1D/α_1B ratio = 79.4). To illustrate the relationship between antagonistic activity and chemical structure, molecular docking studies were performed using the homology models of α₁ receptors. Binding mechanism indicated that small hydrophobic substituents attached to the arylpiperazine moiety were essential for rational design of α_1D-selective antagonists.     

First crystal structure of an endo-inulinase,INU2, from Aspergillus ficuum: Discovery of an extra-pocket in the catalytic domain responsible for its endo-activity

Endo-inulinase is a member of glycosidase hydrolase family 32 (GH32) degrading fructans of the inulin type with an endo-cleavage mode and is an important class of industrial enzyme. In the present study, we report the first crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum at 1.5 Å. It was solved by molecular replacement with the structure of exo-inulinase as search model. The 3D structure presents a bimodular arrangement common to other GH32 enzymes: a N-terminal 5-fold β-propeller catalytic domain with four β-sheets and a C-terminal β-sandwich domain organized in two β-sheets with five β-strands. The structural analysis and comparison with other GH32 enzymes reveal the presence of an extra pocket in the INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)-P-N-G. This cavity would explain the endo-activity of the enzyme, the critical role of Trp40 and particularly the cleavage at the third unit of the inulin(-like) substrates. Crystal structure at 2.1 Å of INU2 complexed with fructosyl molecules, experimental digestion data and molecular modelling studies support these hypotheses.     

The molecular mechanics program DUPLEX: Computing structures of carcinogen modified DNA by surveying the potential energy surface

The nucleic acids molecular mechanics program DUPLEX has been designed with useful features for surveying the potential energy surface of polynucleotides, especially ones that are modified by polycyclic aromatic carcinogens. The program features helpful strategies for addressing the multiple minimum problem: (1) the reduced variable domain of torsion angle space; (2) search strategies that emphasize large scale searches for smaller subunits, followed by building to larger units by a variety of strategies; (3) the use of penalty functions to aid the minimizer in locating selected structural types in first stage minimizations; penalty functions are released in terminal minimizations to yield final unrestrained minimum energy conformations. Predictive capability is illustrated by DNA modified by activated benzo[a]pyrenes.     

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.     

蛋白激酶C同工酶分子结构及功能研究进展

蛋白激酶Ｃ（ＰＫＣ）是至少包括１１种亚型在内的丝／苏氨酸蛋白激酶家族,可分为传统型（ｃＰＫＣｓ）、新型（ｎＰＫＣｓ）、非典型（ａＰＫＣｓ）和ＰＫＣ－ｕ四大类。各ＰＫＣ亚型在ＡＴＰ结合位点、磷脂酰基转移位点、假性底物位点、佛波酯结合位点的氨基酸序列既高度保守又有变异。ＰＫＣ在机体内分布和作用十分广泛,本文主要介绍了ＰＫＣ在肿瘤形成、侵润和转移及肿瘤耐药性产生,调节造血干／祖细胞定向分化成熟,以及激素     

The interface of protein structure, protein biophysics, and molecular evolution

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction.     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.     

Kinetics and molecular modelling studies of the inhibition of protein tyrosine phosphatases by N,N-dimethylhydroxylamine complexes of vanadium(V)

 Bis(N,N-dimethylhydroxamido)hydroxooxovanadate and a derivative complex formed with dithiothreitol have been shown to be excellent inhibitors of the function of two protein tyrosine phosphatases, leucocyte antigen related phosphatase (LAR) and protein tyrosine phosphatase-1B (PTP1B). Inhibition constants of 1.0±0.3 μM and 2.3±0.3 μM, respectively, were determined for the inhibition of LAR by the two complexes. The inhibition of PTP1B is not substantially different. Unlike the structurally related hydrogen peroxide complexes of vanadium, these complexes inhibit in a fully reversible manner that is consistent with a non-oxidative process. Molecular modelling studies suggest the main stabilizing interaction is a cyclic H-bonded structure involving the conserved active site aspartate. Hydrophobic stabilization interactions were also suggested. Received: 17 February 1998 / Accepted: 6 July 1998     

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.