Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II

Oxygenic photosynthesis in plants, algae and cyanobacteria is initiated at photosystem II, a homodimeric multisubunit protein-cofactor complex embedded in the thylakoid membrane. Photosystem II captures sunlight and powers the unique photo-induced oxidation of water to atmospheric oxygen. Crystallographic investigations of cyanobacterial photosystem II have provided several medium-resolution structures (3.8 to 3.2 A) that explain the general arrangement of the protein matrix and cofactors, but do not give a full picture of the complex. Here we describe the most complete cyanobacterial photosystem II structure obtained so far, showing locations of and interactions between 20 protein subunits and 77 cofactors per monomer. Assignment of 11 beta-carotenes yields insights into electron and energy transfer and photo-protection mechanisms in the reaction centre and antenna subunits. The high number of 14 integrally bound lipids reflects the structural and functional importance of these molecules for flexibility within and assembly of photosystem II. A lipophilic pathway is proposed for the diffusion of secondary plastoquinone that transfers redox equivalents from photosystem II to the photosynthetic chain. The structure provides information about the Mn4Ca cluster, where oxidation of water takes place. Our study uncovers near-atomic details necessary to understand the processes that convert light to chemical energy.     

A giant chlorophyll-protein complex induced by iron deficiency in cyanobacteria

Cyanobacteria are abundant throughout most of the world's water bodies and contribute significantly to global primary productivity through oxygenic photosynthesis. This reaction is catalysed by two membrane-bound protein complexes, photosystem I (PSI) and photosystem II (PSII), which both contain chlorophyll-binding subunits functioning as an internal antenna. In addition, phycobilisomes act as peripheral antenna systems, but no additional light-harvesting systems have been found under normal growth conditions. Iron deficiency, which is often the limiting factor for cyanobacterial growth in aquatic ecosystems, leads to the induction of additional proteins such as IsiA (ref. 3). Although IsiA has been implicated in chlorophyll storage, energy absorption and protection against excessive light, its precise molecular function and association to other proteins is unknown. Here we report the purification of a specific PSI-IsiA supercomplex, which is abundant under conditions of iron limitation. Electron microscopy shows that this supercomplex consists of trimeric PSI surrounded by a closed ring of 18 IsiA proteins binding around 180 chlorophyll molecules. We provide a structural characterization of an additional chlorophyll-containing, membrane-integral antenna in a cyanobacterial photosystem.     

Crystal structure of plant photosystem I

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on Earth. The conversion of sunlight into chemical energy is driven by two multisubunit membrane protein complexes named photosystem I and II. We determined the crystal structure of the complete photosystem I (PSI) from a higher plant (Pisum sativum var. alaska) to 4.4 A resolution. Its intricate structure shows 12 core subunits, 4 different light-harvesting membrane proteins (LHCI) assembled in a half-moon shape on one side of the core, 45 transmembrane helices, 167 chlorophylls, 3 Fe-S clusters and 2 phylloquinones. About 20 chlorophylls are positioned in strategic locations in the cleft between LHCI and the core. This structure provides a framework for exploration not only of energy and electron transfer but also of the evolutionary forces that shaped the photosynthetic apparatus of terrestrial plants after the divergence of chloroplasts from marine cyanobacteria one billion years ago.     

Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å

Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 ?. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 ?. From our electron density map, we located all of the metal atoms of the Mn(4)CaO(5) cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn(4)CaO(5) cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.     

Does Q beta replicase synthesize RNA in the absence of template?

Q beta replicase, in the absence of added template, will synthesize RNA autocatalytically. A variety of small RNa species, termed '6S RNAs' are generated. As this reaction purportedly occurs in the absence of template, it has been termed 'de novo' RNA synthesis. The question of whether Q beta replicase can polymerize replicatable RNA molecules, without instruction from a template, has important evolutionary implications. The finding that Q beta replicase was able to synthesize RNA de novo was based on (1) failure to find contaminating RNA in Q beta replicase preparations; (2) differences in the sizes of products of apparently identical reactions; and (3) kinetic differences between template-instructed and de novo reactions. Here wer describe a procedure for production of Q beta replicase lacking one of its subunits, ribosomal protein S1, involving column chromatography in the presence of a low concentration of urea. We show that the resulting highly purified enzyme will not synthesize detectable RNA in the absence of added template. We show also that the ability to perform a reaction kinetically indistinguishable from the de novo synthesis reaction can be restored to the highly purified enzyme by adding a heat-stable, alkali-labile component of Q beta replicase preparations. Thus our findings suggest that, in the novo reaction, Q beta replicase is replicating previously undetected contaminating RNA molecules.     

Protein-only transmission of three yeast prion strains

Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.     

Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide

Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.     

An atypical haem in the cytochrome b(6)f complex

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b(6)f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc(1), cytochrome b(6)f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 A of cytochrome b(6)f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc(1) but also exhibits some unique features, such as binding chlorophyll, beta-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.     

Iron deficiency induces the formation of an antenna ring around trimeric photosystem I in cyanobacteria

Although iron is the fourth most abundant element in the Earth's crust, its concentration in the aquatic ecosystems-particularly the open oceans-is sufficiently low to limit photosynthetic activity and phytoplankton growth. Cyanobacteria, a major class of phytoplankton, respond to iron deficiency by expressing the 'iron-stress-induced' gene, isiA(ref. 3). The protein encoded by this gene has an amino-acid sequence that shows significant homology with one of the chlorophyll a-binding proteins (CP43) of photosystem II (PSII). The precise function of the CP43-like protein, here called CP43', has not been elucidated, although there have been many suggestions. Here we show that CP43' associates with photosystem I (PSI) to form a complex that consists of a ring of 18 CP43' molecules around a PSI trimer. This significantly increases the size of the light-harvesting system of PSI. The utilization of a PSII-like protein as an extra antenna for PSI emphasises the flexibility of cyanobacterial light-harvesting systems, and seems to be a strategy which compensates for the lowering of phycobilisome and PSI levels in response to iron deficiency.     

Low-light-adapted Prochlorococcus species possess specific antennae for each photosystem

Prochlorococcus, the most abundant genus of photosynthetic organisms, owes its remarkably large depth distribution in the oceans to the occurrence of distinct genotypes adapted to either low- or high-light niches. The pcb genes, encoding the major chlorophyll-binding, light-harvesting antenna proteins in this genus, are present in multiple copies in low-light strains but as a single copy in high-light strains. The basis of this differentiation, however, has remained obscure. Here we show that the moderate low-light-adapted strain Prochlorococcus sp. MIT 9313 has one iron-stress-induced pcb gene encoding an antenna protein serving photosystem I (PSI)--comparable to isiA genes from cyanobacteria--and a constitutively expressed pcb gene encoding a photosystem II (PSII) antenna protein. By comparison, the very low-light-adapted strain SS120 has seven pcb genes encoding constitutive PSI and PSII antennae, plus one PSI iron-regulated pcb gene, whereas the high-light-adapted strain MED4 has only a constitutive PSII antenna. Thus, it seems that the adaptation of Prochlorococcus to low light environments has triggered a multiplication and specialization of Pcb proteins comparable to that found for Cab proteins in plants and green algae.     

Macromolecular structural transitions in Pf1 filamentous bacterial virus.

The filamentous bacterial virus Pf1 is a simple model for biological filaments. We have studied the structure of the virion and report here that the helix parameters of Pf1 change sharply with temperature at about 8 degrees C. Local interactions between protein subunits change by only a few tenths of an angstrom, but the changes are amplified between one end and the other of the virion to a rotation of 15 turns and a translation of 1,000 A. The limited nature of the phase transition is probably due to the constraints of 'knobs-into-holes' interaction between side chains of adjacent alpha-helical protein subunits. Treatment of the virion with ether causes a rearrangement of protein subunits into sheets, with the alpha-helices normal to the plane of the sheet. This phase transition suggests a model for virion assembly in the bacterial membrane.     

明渠湍流中的主要相干结构模式

利用自主开发的高帧频明渠湍流粒子图像测速(PIV)系统测量了3种Reynolds数下的恒定均匀流时间序列流场,运用本征正交分解能够给出数据集的最优模态的特性,分析了明渠湍流的主要相干结构模式及其能量关系。湍动能主要集中在少数几个低阶模态上,第1阶模态含有约30%以上的湍动能。Q2(Q4)事件是明渠湍流中除平均流动外含能最多、最主要的大尺度结构,湍动能主要通过Q2(Q4)事件向其他结构传递。它并不完全是发夹涡群诱导的结构,相反从能量的角度来看,发夹涡群的产生与维持与它有关键关系。Q2(Q4)事件可能存在大范围独立维持的大尺度机制。同时本征正交分解(POD)技术成功提取了发夹涡群结构,流向范围达到约3.4h,垂向尺度从交叠层一直到水面附近,整个结构与壁面呈约10°夹角。发夹涡群载有约23%的平均湍动能,并主要集中在处于对数区的中间部分。其他含能较少的大尺度结构有水面涡旋、单双涡结构、独立涡包等。     

溶剂法合成聚琥珀酰亚胺

以L-天冬氨酸为原料,采用溶剂法合成聚琥珀酰亚胺,并研究了催化剂、反应时间和混和溶剂配比对反应的影响．用凝胶渗透色谱测定了聚琥珀酰亚胺的相对分子质量,并用FTIR和^1H NMR对其进行了结构表征．     

Structural basis of protein phosphatase 1 regulation

The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes. The human genome encodes a far greater number of Ser/Thr protein kinases than of phosphatases. Protein phosphatase 1 (PP1), in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell-cycle progression and muscle relaxation. PP1 has evolved effective catalytic machinery but lacks substrate specificity. Substrate specificity is conferred upon PP1 through interactions with a large number of regulatory subunits. The regulatory subunits are generally unrelated, but most possess the RVxF motif, a canonical PP1-binding sequence. Here we reveal the crystal structure at 2.7 A resolution of the complex between PP1 and a 34-kDa N-terminal domain of the myosin phosphatase targeting subunit MYPT1. MYPT1 is the protein that regulates PP1 function in smooth muscle relaxation. Structural elements amino- and carboxy-terminal to the RVxF motif of MYPT1 are positioned in a way that leads to a pronounced reshaping of the catalytic cleft of PP1, contributing to the increased myosin specificity of this complex. The structure has general implications for the control of PP1 activity by other regulatory subunits.     

SAP-controlled T-B cell interactions underlie germinal centre formation

Generation of long-term antibody-mediated immunity depends on the germinal centre reaction, which requires cooperation between antigen-specific T and B lymphocytes. In human X-linked lymphoproliferative disease and its gene-targeted mouse model, loss-of-function mutations in signalling lymphocyte activation molecule-associated protein (SAP, encoded by SH2D1a) cause a profound defect in germinal centre formation by an as yet unknown mechanism. Here, using two-photon intravital imaging, we show that SAP deficiency selectively impairs the ability of CD4(+) T cells to stably interact with cognate B cells but not antigen-presenting dendritic cells. This selective defect results in a failure of antigen-specific B cells to receive adequate levels of contact-dependent T-cell help to expand normally, despite Sap(-/-) T cells exhibiting the known characteristics of otherwise competent helper T cells. Furthermore, the lack of stable interactions with B cells renders Sap(-/-) T cells unable to be efficiently recruited to and retained in a nascent germinal centre to sustain the germinal centre reaction. These results offer an explanation for the germinal centre defect due to SAP deficiency and provide new insights into the bi-directional communication between cognate T and B cells in vivo.     

长江三峡库区第四纪哺乳动物群及生态环境的演替

根据长江三峡库区范围已发现的２８个第四纪哺乳动物化石点资料,应用系统分析法,对其形成时代反复论证后,通过性质比较,确认该区第四纪哺乳动物群性质为热带－亚热带森林为主的东洋界大熊猫动物群,其演替过程可分为５个阶段。其演替原因是环境的变化,主导因素是造貌运动和气候变化,但到中晚全新世则为人类活动。     

Three-dimensional structure of the bacterial protein-translocation complex SecYEG

Transport and membrane integration of polypeptides is carried out by specific protein complexes in the membranes of all living cells. The Sec transport path provides an essential and ubiquitous route for protein translocation. In the bacterial cytoplasmic membrane, the channel is formed by oligomers of a heterotrimeric membrane protein complex consisting of subunits SecY, SecE and SecG. In the endoplasmic reticulum membrane, the channel is formed from the related Sec61 complex. Here we report the structure of the Escherichia coli SecYEG assembly at an in-plane resolution of 8 A. The three-dimensional map, calculated from two-dimensional SecYEG crystals, reveals a sandwich of two membranes interacting through the extensive cytoplasmic domains. Each membrane is composed of dimers of SecYEG. The monomeric complex contains 15 transmembrane helices. In the centre of the dimer we observe a 16 x 25 A cavity closed on the periplasmic side by two highly tilted transmembrane helices. This may represent the closed state of the protein-conducting channel.     

Structure of fumarate reductase from Wolinella succinogenes at 2.2 A resolution

Fumarate reductase couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalysed by the related complex II of the respiratory chain (succinate dehydrogenase). Here we describe the crystal structure at 2.2 A resolution of the three protein subunits containing fumarate reductase from the anaerobic bacterium Wolinella succinogenes. Subunit A contains the site of fumarate reduction and a covalently bound flavin adenine dinucleotide prosthetic group. Subunit B contains three iron-sulphur centres. The menaquinol-oxidizing subunit C consists of five membrane-spanning, primarily helical segments and binds two haem b molecules. On the basis of the structure, we propose a pathway of electron transfer from the dihaem cytochrome b to the site of fumarate reduction and a mechanism of fumarate reduction. The relative orientations of the soluble and membrane-embedded subunits of succinate:quinone oxidoreductases appear to be unique.     

Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy

Voltage-gated ion channels are transmembrane proteins that are essential for nerve impulses and regulate ion flow across cell membranes in response to changes in membrane potential. They are made up of four homologous domains or subunits, each of which contains six transmembrane segments. Studies of potassium channels have shown that the second (S2) and fourth (S4) segments contain several charged residues, which sense changes in voltage and form part of the voltage sensor. Although these regions clearly undergo conformational changes in response to voltage, little is known about the nature of these changes because voltage-dependent distance changes have not been measured. Here we use lanthanide-based resonance energy transfer to measure distances between Shaker potassium channel subunits at specific residues. Voltage-dependent distance changes of up to 3.2 A were measured at several sites near the S4 segment. These movements directly correlated with electrical measurements of the voltage sensor, establishing the link between physical changes and electrical charge movement. Measured distance changes suggest that the region associated with the S4 segment undergoes a rotation and possible tilt, rather than a large transmembrane movement, in response to voltage. These results demonstrate the first in situ measurement of atomic scale movement in a trans-membrane protein.