Biomedical subjects

James Barber

Publications and source records attributed to James Barber.

At least 37 records · Page 2Linked to original sources

PubMed2004

Engine of life and big bang of evolution: a personal perspective.

Photosystem II (PS II) is the engine for essentially all life on our planet and its beginning 2.5 billion years ago was the 'big bang of evolution.' It produces reducing equivalents for making organic compounds on an enormous scale and at the same time provides us with an oxygenic atmosphere and protection against UV radiation (in the form of the ozone layer). In 1967, when I began my career in photosynthesis research, little was known about PS II. The Z-scheme had been formulated [Hill and Bendall (1960) Nature 186: 136-137] and Boardman and Anderson [(1964) Nature 203: 166-167] had isolated PS II as a discrete biochemical entity. PS II was known not only to be the source of oxygen but of variable chlorophyll fluorescence [Duysens and Sweers (1963) In: Studies on Microalgae and Photosynthetic Bacteria, pp. 353-372. University of Tokyo Press, Tokyo] and delayed chlorophyll fluorescence [Arnold and Davidson (1954) J Gen Physiol 37: 677-684]. P680 had just been discovered [Döring et al. (1967) Z Naturforsch 22b: 639-644]. No wonder the 'black box of PS II' was described at that time by Bessel Kok and George Cheniae [Current Topics in Bioenergetics 1: 1-47 (1966)] as the 'inner sanctum of photosynthesis.' What a change in our level of understanding of PS II since then! The contributions of many talented scientists have unraveled the mechanisms and structural basis of PS II function and we are now very close to revealing the molecular details of the remarkable and thermodynamically demanding reaction which it catalyzes, namely the splitting of water into its elemental constituents. It has been a privilege to be involved in this journey.

Journal Article↗

PubMed2004

Analysis of the Structure of the PsbO Protein and its Implications.

The PsbO protein is a ubiquitous extrinsic subunit of Photosystem II (PS II), the water splitting enzyme of photosynthesis. A recently determined 3D X-ray structure of a cyanobacterial protein bound to PS II has given an opportunity to conduct complete analyses of its sequence and structural characteristics using bioinformatic methods. Multiple sequence alignments for the PsbO family are constructed and correlated with the cyanobacterial structure. We identify the most conserved regions of PsbO and the mapping of their positions within the structure indicates their functional roles especially in relation to interactions of this protein with the lumenal surface of PS II. Homologous models for eukaryotic PsbO were built in order to compare with the prokaryotic protein. We also explore structural homology between PsbO and other proteins for which 3D structures are known and determine its structural classification. These analyses contribute to the understanding of the function and evolutionary origin of the PS II manganese stabilising protein.

Journal Article↗

PubMed2003

Characterization of clonogenic multiple myeloma cells.

The identity of the cells responsible for the initiation and maintenance of multiple myeloma (MM) remains unclear largely because of the difficulty growing MM cells in vitro and in vivo. MM cell lines and clinical specimens are characterized by malignant plasma cells that express the cell surface antigen syndecan-1 (CD138); however, CD138 expression is limited to terminally differentiated plasma cells during B-cell development. Moreover, circulating B cells that are clonally related to MM plasma cells have been reported in some patients with MM. We found that human MM cell lines contained small (< 5%) subpopulations that lacked CD138 expression and had greater clonogenic potential in vitro than corresponding CD138+ plasma cells. CD138- cells from clinical MM samples were similarly clonogenic both in vitro and in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, whereas CD138+ cells were not. Furthermore, CD138- cells from both cell lines and clinical samples phenotypically resembled postgerminal center B cells, and their clonogenic growth was inhibited by the anti-CD20 monoclonal antibody rituximab. These data suggest that MM "stem cells" are CD138- B cells with the ability to replicate and subsequently differentiate into malignant CD138+ plasma cells.

Animals↗

PubMed2003

Structure of a photosystem II supercomplex isolated from Prochloron didemni retaining its chlorophyll a/b light-harvesting system.

Prochlorophytes are a class of cyanobacteria that do not use phycobiliproteins as light-harvesting systems, but contain chlorophyll (Chl) a/b-binding Pcb proteins. Recently it was shown that Pcb proteins form an 18-subunit light-harvesting antenna ring around the photosystem I (PSI) trimeric reaction center complex of the prochlorophyte Prochlorococcus marinus SS120. Here we have investigated whether the symbiotic prochlorophyte Prochloron didemni also contains the same supermolecular complex. Using cells isolated directly from its ascidian host, we found no evidence for the presence of the Pcb-PSI supercomplex. Instead we have identified and characterized a supercomplex composed of photosystem II (PSII) and Pcb proteins. We show that 10-Pcb subunits associate with the PSII dimeric reaction center core to form a giant complex having an estimated Mr of 1,500 kDa with dimensions of 210 x 290 A. Five-Pcb subunits flank each long side of the dimer and assuming each binds 13 Chl molecules, increase the antenna size of PSII by approximately 200%. Fluorescence emission studies indicate that energy transfer occurs efficiently from the Pcb antenna. Modeling using the x-ray structure of cyanobacterial PSII suggests that energy transfer to the PSII reaction center is via the Chls bound to the CP47 and CP43 proteins.

Light-Harvesting Protein Complexes↗

PubMed2003

Exploring the ability of chlorophyll b to bind to the CP43' protein induced under iron deprivation in a mutant of Synechocystis PCC 6803 containing the cao gene.

Cyanobacteria, unlike plants and green algae, do not contain chlorophyll (Chl) b. This is because of the absence of the cao gene which encodes the enzyme that catalyses a two step oxygenation of chlorophyllide a to chlorophyllide b. Recently, however, the cao gene of higher plants was engineered into Synechocystis PCC 6803 leading to Chl b synthesis in this cyanobacterium [Satoh et al., J. Biol. Chem. 276 (2001) 4293-4297]. Here we use this same cao-plus mutant to show that Chl b can bind to the CP43' protein, expressed in cells exposed to low iron levels, which normally binds Chl a only. In so doing CP43' is changed to a Chl a/Chl b-binding protein and in this respect resembles the closely related Chl a/Chl b-binding Pcb protein of prochlorophytes (green oxyphotobacteria). The results emphasise the possibility of using an in vitro system to elucidate factors which control the binding of these two different forms of chlorophylls to the six transmembrane helical light-harvesting proteins of oxygenic photosynthetic organisms.

Bacterial Proteins↗

PubMed2003

Time-resolved absorption and emission show that the CP43' antenna ring of iron-stressed synechocystis sp. PCC6803 is efficiently coupled to the photosystem I reaction center core.

Excitation energy transfer and trapping processes in an iron stress-induced supercomplex of photosystem I from the cyanobacterium Synechocystis sp. PCC6803 were studied by time-resolved absorption and fluorescence spectroscopy on femtosecond and picosecond time scales. The data provide evidence that the energy transfer dynamics of the CP43'-PSI supercomplex are consistent with energy transfer processes that occur in the Chl a network of the PSI trimer antenna. The most significant absorbance changes in the CP43'-PSI supercomplex are observed within the first several picoseconds after the excitation into the spectral region of CP43' absorption (665 nm). The difference time-resolved spectra (DeltaDeltaA) resulting from subtraction of the PSI trimer kinetic data from the CP43'-PSI supercomplex data indicate three energy transfer processes with time constants of 0.2, 1.7, and 10 ps. The 0.2 ps kinetic phase is tentatively interpreted as arising from energy transfer processes originating within or between the CP43' complexes. The 1.7 ps phase is interpreted as possibly arising from energy transfer from the CP43' ring to the PSI trimer via closely located clusters of Chl a in CP43' and the PSI core, while the slower 10 ps process might reflect the overall excitation transfer from the CP43' ring to the PSI trimer. These three fast kinetic phases are followed by a 40 ps overall excitation decay in the supercomplex, in contrast to a 25 ps overall decay observed in the trimer complex without CP43'. Excitation of Chl a in both the CP43'-PSI antenna supercomplex and the PSI trimer completely decays within 100 ps, resulting in the formation of P700(+). The data indicate that there is a rapid and efficient energy transfer between the outer antenna ring and the PSI reaction center complex.

Bacterial Proteins↗

PubMed2003

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.

Cryoelectron Microscopy↗

PubMed2003

Using rational screening and electron microscopy to optimize the crystallization of succinate:ubiquinone oxidoreductase from Escherichia coli.

The membrane-bound respiratory complex II, succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli, has been anaerobically expressed, then purified and crystallized. The initial crystals obtained were small and diffracted poorly. In order to facilitate structure determination, rational screening and sample-quality analysis using electron microscopy was implemented. The crystals of SQR from E. coli belong to the trigonal space group R32, with unit-cell parameters a = b = 138.7, c = 521.9 A, and diffract to 2.6 A resolution. The optimization strategy used for obtaining well diffracting SQR crystals is applicable to a wide range of membrane proteins.

Anisotropy↗

PubMed2003

Three-dimensional reconstruction of a light-harvesting complex I-photosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii. Insights into light harvesting for PSI.

A supercomplex containing the photosystem I (PSI) and chlorophyll a/b light-harvesting complex I (LHCI) has been isolated using a His-tagged mutant of Chlamydomonas reinhardtii. This LHCI-PSI supercomplex contained approximately 215 chlorophyll molecules of which 175 were estimated to be chlorophyll a and 40 to be chlorophyll b, based on P700 oxidation and chlorophyll a/b ratio measurements. Its room temperature long wavelength absorption peak was at 680 nm, and it emitted chlorophyll fluorescence maximally at 715 nm (77 K). The LHCI was composed of four or more different types of Lhca polypeptides including Lhca3. No LHCII proteins or other phosphoproteins were detected in the LHCI-PSI supercomplexes suggesting that the cells from which they were isolated were in State 1. Electron microscopy of negatively stained samples followed by image analysis revealed the LHCI-PSI supercomplex to have maximal dimensions of 220 A by 180 A and to be approximately 105 A thick. An averaged top view was used to model in x-ray and electron crystallographic data for PSI and Lhca proteins respectively. We conclude that the supercomplex consists of a PSI reaction center monomer with 11 Lhca proteins arranged along the side where the PSI proteins, PsaK, PsaJ, PsaF, and PsaG are located. The estimated molecular mass for the complex is 700 kDa including the bound chlorophyll molecules. The assignment of 11 Lhca proteins is consistent with a total chlorophyll level of 215 assuming that the PSI reaction center core binds approximately 100 chlorophylls and that each Lhca subunit binds 10 chlorophylls. There was no evidence for oligomerization of Chlamydomonas PSI in contrast to the trimerization of PSI in cyanobacteria.

Animals↗

PubMed2003

Oxidation of the two beta-carotene molecules in the photosystem II reaction center.

We present a spectroscopic characterization of the two nonequivalent beta-carotene molecules in the photosystem II reaction center. Their electronic and vibrational properties exhibit significant differences, reflecting a somewhat different configuration for these two cofactors. Both carotenoid molecules are redox-active and can be oxidized by illumination of the reaction centers in the presence of an electron acceptor. The radical cation species show similar differences in their spectroscopic properties. The results are discussed in terms of the structure and unusual function of these carotenoids. In addition, the attribution of resonance Raman spectra of photosystem II preparations excited in the range 800-900 nm is discussed. Although contributions of chlorophyll cations cannot be formally ruled out, our results demonstrate that these spectra mainly arise from the cation radical species of the two carotenoids present in photosystem II reaction centers.

Cations↗

PubMed2003

Length, time, and energy scales of photosystems.

The design of photosynthetic systems reflects the length scales of the fundamental physical processes. Energy transfer is rapid at the few angstrom scale and continues to be rapid even at the 50-A scale of the membrane thickness. Electron tunneling is nearly as rapid at the shortest distances, but becomes physiologically too slow well before 20 A. Diffusion, which starts out at a relatively slow nanosecond time scale, has the most modest slowing with distance and is physiologically competent at all biologically relevant distances. Proton transfer always operates on the shortest angstrom scale. The structural consequences of these distance dependencies are that energy transfer networks can extend over large, multisubunit and multicomplex distances and take leaps of 20 A before entering the domain of charge separating centers. Electron transfer systems are effectively limited to individual distances of 15 A or less and span the 50 A dimensions of the bioenergetic membrane by use of redox chains. Diffusion processes are generally used to cover the intercomplex electron transfer distances of 50 A and greater and tend to compensate for the lack of directionality by restricting the diffusional space to the membrane or the membrane surface, and by multiplying the diffusing species through the use of pools. Proton transfer reactions act over distances larger than a few angstroms through the use of clusters or relays, which sometimes rely on water molecules and which may only be dynamically assembled. Proteins appear to place a premium on robustness of design, which is relatively easily achieved in the long-distance physical processes of energy transfer and electron tunneling. By placing cofactors close enough, the physical process is relatively rapid compared to decay processes. Thus suboptimal conditions such as cofactor orientation, energy level, or redox potential level can be tolerated and generally do not have to be finely tuned. The most fragile regions of design tend to come in areas of complex formation and catalysis involving proton management, where relatively small changes in distance or mutations can lead to a dramatic decrease in turnover, which may already be limiting the overall speed of energy conversion in these proteins. Light-activated systems also face a challenge to robust function from the ever-present dangers of high redox potential chemistry. This can turn the protein matrix and wandering oxygen molecules into unintentional redox partners, which in the case of PSII requires the frequent, costly replacement of protein subunits.

Bacterial Proteins↗

PubMed2003

The 1.45 A three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus.

The conversion of solar radiation to chemical energy by photosynthetic organisms provides the primary driving force for life on earth. Light energy is captured by a variety of pigments, usually bound to proteins, which vary with different types of organisms. We report here the 1.45 A resolution three-dimensional structure of one such pigment protein, C-phycocyanin, from Synechococcus elongatus. The structure is at the highest resolution achieved for any such phycobiliprotein. This level of resolution was made possible by implementing a novel crystallization method whereby nucleation is decoupled from subsequent growth, by incubating crystallizing drops for 7h under nucleation conditions and then transferring them to metastable conditions for growth. This is done without touching the crystallization drops throughout the process.

Crystallography, X-Ray↗

PubMed2003

Photosystem II: the engine of life.

Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3.7/3.8 A, have allowed assignments to be made to the redox active cofactors involved in the light-driven water-plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the DI protein and approximately 7 A from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.

Chlorophyll↗

PubMed2003

Interaction of the allophycocyanin core complex with photosystem II.

Allophycocyanin core complexes were purified from the thermophilic cyanobacterium Thermosynechococcus elongatus and analysed by negative-stain electron microscopy and single-particle averaging. The purified complex was found to consist of three allophycocyanin cylinders. The single-particle analysis of end-on views of the complex revealed a mirror axis, indicative of two-fold symmetry. This observation allowed the assignment of the allophycocyanin base cylinders and the identification of their potential interaction sites with the thylakoid membrane and with the photosystem II reaction centre in particular. The T. elongatus allophycocyanin core projection map, together with published information on the structure of photosystem II for the same organism, was used to construct a model for the allophycocyanin core-photosystem II dimer supercomplex, from which docking sites between both complexes are suggested. The implications of this association for energy transfer from allophycocyanin to photosystem II are discussed.

Journal Article↗

PubMed2003

Anti-tumour activity of interferon-alpha in multiple myeloma: role of interleukin 6 and tumor cell differentiation.

Interferon-alpha (IFN-alpha) is a pleotropic cytokine that has clinical activity against a wide variety of malignancies, including multiple myeloma (MM). In vitro, IFN-alpha has diverse effects on both normal and malignant cells, however, the exact mechanisms responsible for its clinical anti-tumour activity remain unclear. We found that IFN-alpha inhibited MM cell proliferation in association with cell cycle arrest at G1 and limited the clonogenic growth of both MM cell lines and primary patient specimens. At the doses tested, IFN-alpha was not cytotoxic, but induced terminal plasma cell differentiation resulting in the loss of clonogenicity. These activities were markedly enhanced by the major MM growth factor interleukin 6 (IL-6). Moreover, IL-6 was required for this process, as neutralizing antibodies against IL-6 inhibited the effects of IFN-alpha. IL-6 also induced MM cell terminal differentiation when combined with a second, unrelated, antiproliferative agent bryostatin-1, suggesting that its differentiating activities are preferentially enhanced in the presence of agents that inhibit cell cycling. These results suggest that the differentiating activities of IFN-alpha may play a role in its clinical antimyeloma activity and provide the rationale for clinical differentiation therapy in MM.

Analysis of Variance↗

PubMed2002

Photosystem II: a multisubunit membrane protein that oxidises water.

A structure of photosystem II recently determined by X-ray crystallography at 3.8 A resolution complements structural studies using high-resolution electron microscopy and represents a major step towards understanding how photosynthetic organisms use light energy to oxidise water.

Chlorophyll↗

PubMed2002

P680: what is it and where is it?

Electron and X-ray crystallography have provided intermediate structural models for photosystem II (PSII), the membrane located multisubunit complex which uses light energy to split water into its elemental constituents. This reaction is thermodynamically demanding and involves the production of redox potentials in excess of 1 V. Structural analyses have now shown that the primary oxidant, P680, is not a 'special pair' of chlorophylls, as in other types of photosynthetic reaction centres, but a tetramer of equally spaced chlorophyll a molecules. Its high redox potential, and the involvement of four weakly coupled isoenergetic monomers rather than a strongly excitonically coupled 'special pair', has implications for redox mechanisms which are unique to PSII, and therefore not found in any other photosynthetic system. The importance of these features is discussed.

Crystallography, X-Ray↗

PubMed1997

STRUCTURE AND MEMBRANE ORGANIZATION OF PHOTOSYSTEM II IN GREEN PLANTS.

Photosystem II (PSII) is the pigment protein complex embedded in the thylakoid membrane of higher plants, algae, and cyanobacteria that uses solar energy to drive the photosynthetic water-splitting reaction. This chapter reviews the primary, secondary, tertiary, and quaternary structures of PSII as well as the function of its constituent subunits. The understanding of in vivo organization of PSII is based in part on freeze-etched and freeze-fracture images of thylakoid membranes. These images show a resolution of about 40-50 A and so provide information mainly on the localization, heterogeneity, dimensions, and shapes of membrane-embedded PSII complexes. Higher resolution of about 15-40 A has been obtained from single particle images of isolated PSII complexes of defined and differing subunit composition and from electron crystallography of 2-D crystals. Observations are discussed in terms of the oligomeric state and subunit organization of PSII and its antenna components.

Journal Article↗