RESUMEN
The light-harvesting chlorophyll a/b complex (LHC-II) found in green plants has at least three functions: it absorbs light energy for transfer to the reaction centers, it is involved in keeping the photosynthetic membranes stacked, and it regulates energy distribution between the two photosystems. We have developed a procedure to produce large vesicles consisting almost exclusively of two-dimensional crystalline domains of LHC-II in which LHC-II is biochemically and structurally intact, as shown by SDS-PAGE, response to cations, and 77K fluorescence excitation spectra. The vesicles were examined by cryoelectron microscopy and analyzed, in projection, to a resolution of 17 A. Their surface lattice consists of trimers arranged in interlocking circles; the two-sided plane group is p321 (unit cell dimension, a = 124 A) with two, oppositely facing trimers/unit cell. Individual trimers consist of matter arranged in a ring, around a central cavity, an appearance similar to that obtained in some conditions using negative stain (Li, J., 1985. Proc. Natl. Acad. Sci. USA. 82:386-390). The monomer (approximately 45 x 20 A) is seen as two domains of slightly different size at this resolution. The thickness of single layers is approximately 48 A, measured from edge-on views of the frozen vesicles. Based on these dimensions, the molecular mass of the monomer is approximately 30 kD. Therefore, each monomer appears to be composed of a single polypeptide and its associated pigments.
Asunto(s)
Clorofila/análisis , Proteínas de Plantas/análisis , Clorofila/aislamiento & purificación , Cristalización , Electroforesis en Gel de Poliacrilamida , Congelación , Procesamiento de Imagen Asistido por Computador , Complejos de Proteína Captadores de Luz , Microscopía Electrónica , Microscopía Fluorescente , Proteínas del Complejo del Centro de Reacción Fotosintética , Proteínas de Plantas/aislamiento & purificación , Espectrometría de Fluorescencia , Coloración y EtiquetadoRESUMEN
We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.
Asunto(s)
Clorofila , Cloroplastos/ultraestructura , Proteínas de Plantas , Cloroplastos/efectos de los fármacos , Cristalización , Fabaceae , Técnica de Fractura por Congelación , Membranas Intracelulares/ultraestructura , Complejos de Proteína Captadores de Luz , Sustancias Macromoleculares , Microscopía Electrónica , Octoxinol , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética , Complejo de Proteína del Fotosistema I , Complejo de Proteína del Fotosistema II , Plantas Medicinales , Polietilenglicoles/farmacología , Espectrometría de FluorescenciaRESUMEN
Photosystem II (PS II) is a photosynthetic reaction center found in higher plants which has the unique ability to evolve oxygen from water. Several groups have formed two-dimensional PS II crystals or have isolated PS II complexes and studied them by electron microscopy and image analysis. The majority of these specimens have not been well characterized biochemically and have yielded relatively low resolution two-dimensional projection maps with a variety of unit cell sizes. We report the characterization of the polypeptide and lipid content of tubular crystals of PS II. The crystals contain the reaction center core polypeptides D1, D2, cytochrome b559, as well as the chlorophyll-binding polypeptides (CP) CP47, CP43, CP29, CP26, CP24, and CP22. The lipid composition was similar to the lipids found in the stacked portion of thylakoids. We also report a 2.0-nm resolution projection map determined by electron microscopy and image analysis of frozen, hydrated PS II crystals. This projection map includes information on the portion of the complex buried in the lipid bilayer. The unit cell is a dimer with unit vectors of 17.0 and 11.4 nm separated by an angle of 106.6 degrees. In addition, Fab fragments against D1 and cytochrome b559 were used to localize those two polypeptides, and thus the reaction center, within the PS II complex. The results indicate that D1 and cytochrome b559 are found within one of the heaviest densities of the monomeric unit.
Asunto(s)
Cloroplastos/química , Grupo Citocromo b/aislamiento & purificación , Proteínas de la Membrana/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/aislamiento & purificación , Criopreservación , Cristalografía , Procesamiento de Imagen Asistido por Computador , Membranas Intracelulares/química , Complejos de Proteína Captadores de Luz , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/ultraestructura , Microscopía Electrónica , Coloración Negativa , Proteínas del Complejo del Centro de Reacción Fotosintética/inmunología , Proteínas del Complejo del Centro de Reacción Fotosintética/ultraestructura , Complejo de Proteína del Fotosistema II , Spinacia oleracea/químicaRESUMEN
Three types of photosystem II (PS II) crystals have been produced using a variety of detergents. Intermediate stages of crystal formation were examined and it was determined that each crystal probably originates from a single grana membrane. Each crystal type was examined by electron microscopy and image processing, providing three different projection maps. The highest resolution results came from type 1 and type 2 crystals. Projection maps from these crystals were examined for two-fold symmetry via difference maps between the unsymmetrized averages and their 180 degrees rotation. A comparison of the final maps shows a high degree of two-fold symmetry, with only slight differences noted in the low density regions of the two halves of the structure. The interpretation is that PS II is a dimer, with the further suggestion that the two reaction center cores may have slightly different complements of antennae polypeptides.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Cristalización , Dimerización , Complejo de Proteína del Fotosistema IIRESUMEN
A consecutive series of ultrathin sections through the distal one-third of a Hydra tentacle has revealed at least four categories of nematocytes: (1) normal, mounted nematocytes, in specific arrangements within the battery cells; (2) degenerating nematocytes, within the battery cells; (3) mature nematocytes, enclosed within endodermal cells: (4) a mature nematocyte, in the enteric cavity. The degenerating nematocytes within the battery cells and the nematocytes in the endoderm and enteric cavity appeared to be aging nematocytes undergoing death and removal. The results provide the first ultrastructural evidence for nematocyte degeneration within battery cells and also suggest phagocytosis of mature nematocytes by endodermal cells.
Asunto(s)
Hydra/citología , Animales , Supervivencia Celular , Endodermo/citología , Uniones Intercelulares/ultraestructura , Microscopía Electrónica , FagocitosisRESUMEN
Different fractions of a mixture of protein-pigment complexes have been separated from one another by non-denaturing gel electrophoresis. These complexes were prepared for observation by inserting electron microscope grids directly into the focused bands of the pigment-protein complexes and resuming electrophoresis for a brief time, so that the complexes were deposited onto the grids. It was found that complexes deposited from each band exhibited distinctly different appearances. It was also found that the exact conditions of electrophoretic deposition onto the grids affected the appearance of the complexes. The protein-pigment complexes were characterized additionally by spectroscopy and denaturing gel electrophoresis.
Asunto(s)
Clorofila/química , Electroforesis en Gel de Agar/métodos , Microscopía Electrónica/métodos , Proteínas del Complejo del Centro de Reacción Fotosintética/ultraestructura , Cloroplastos/química , Complejos de Proteína Captadores de Luz , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Espectrofotometría , Spinacia oleracea/químicaRESUMEN
Ultrastructural and light microscopic observations on the organization of thick and thin regions of hydra's tentacles, made on serial sections and on whole fixed, plastic-embedded tentacles, reveal the existence of two levels of anatomical order in the tentacle ectoderm: (1) The battery-cell complex (BCC), composed of a single epitheliomuscular cell (EMC) and its content of enclosed nematocytes and neurons; and (2) the battery cell complex ring (BCC ring), an arrangement of 4 or more BCCs into larger units organized as rings around the circumference of the tentacle. All EMCs of the distal tentacle appear to contain batteries of nematocytes, and are, therefore, called "battery cells." Apart from battery cell complexes and migrating nematocytes, there are no other cell types in the tentacle ectoderm. Battery cells are composed of three distinct regions: the cell body, peripheral attenuated extensions and myonemes. Thick tentacle bands are composed of cell bodies, whereas thin bands are made up of attenuated extensions. Myonemes contribute to both thick and thin regions. It was confirmed that each battery cell has several myonemes, which appear to interdigitate with myonemes of other more proximal and distal battery cells, but not with battery cells of the same BCC ring. Nematocytes have several basal processes. Some processes insert between myonemes and contact the mesoglea; other processes insert into cuplike extensions of myonemes, and are connected to myonemal cups by desmosomal junctions. These observations are discussed in relation to mechanical and electrical aspects of tentacular contraction and bending.
RESUMEN
Photosystem II (PS II), a eukaryotic photosynthetic reaction center which converts solar energy to chemical energy, is also capable of evolving oxygen, making it uniquely important for our biosphere. We report the formation of two-dimensional crystals of the PS II complex. The crystals were tubular, approximately 0.2 by 1-2 microns. Characterization of the crystals by gel electrophoresis, immunoblotting, and absorption spectroscopy suggested that the crystals contain PS II exclusively, with no other protein complexes. Atomic force microscopy revealed that the complexes were closely packed, suggesting that the process of crystallization involves partial removal of lipid from the membrane. The structure of the complex was investigated using low-dose electron microscopy and image analysis. A projection map at 1.7 nm resolution was produced. The unit cell was 11.5 x 16.1 nm and consisted of two monomeric units arranged around a central cavity to form a dimer. Volume calculations suggested that each dimer consisted of two PS II complexes. The monomeric unit, which appears to be a single PS II complex, had four areas of density. The gap between the two PS II complexes was quite small, indicating that there may be a functional connection between the two halves of the dimer.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/ultraestructura , Cristalización , Procesamiento de Imagen Asistido por Computador , Microscopía/métodos , Microscopía Electrónica , Coloración Negativa , Proteínas del Complejo del Centro de Reacción Fotosintética/aislamiento & purificación , Complejo de Proteína del Fotosistema IIRESUMEN
Photosystem II (PS II), found in the photosynthetic membranes of plants, has the unique ability to split water, evolving atmospheric oxygen as a byproduct. In the photosystem I deficient barley mutant, viridis zb63, PS II is found in 2D crystals but has normal activity [D. Simpson, (1983) Eur. J. Cell Biol. 31, 305-314]. We have isolated these PS II crystals from the mutants and obtained a projection map at 2.0 nm resolution. This map was compared to a projection map of PS II in crystals derived from spinach. The unit cell for the barley crystal was 16.1 x 24.1 nm; for spinach, the unit cell was 11.9 x 16.6 nm. In both cases, there was p2 symmetry and each half of the unit cell included five subareas. After isolation, the barley crystals were unstable, suggesting that, in this case, interactions across membranes within the grana are required to retain ordering of PS II. A comparison of the estimated masses within the PS II dimer from each species indicated that the two crystals probably did not contain the same complement of polypeptides, suggesting that PS II is labile. Nevertheless, the projection maps contained similar structural features, suggesting that PS II lability is restricted and that there is an underlying stable structure.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Cristalización , Hordeum , Modelos Moleculares , Complejo de Proteína del Fotosistema I , Complejo de Proteína del Fotosistema II , Conformación Proteica , Spinacia oleraceaRESUMEN
Phosphorylation of the polyomavirus major capsid protein VP1 plays a role in virus assembly and may function in virus-cell recognition. Previous mapping of the in vivo phosphorylation sites on VP1 identified phosphorylation of threonine residues Thr-63 and Thr-156 (Li, M., and Garcea, R. L. (1994) J. Virol. 68, 320-327). Phosphoserine was detected in a tryptic phosphopeptide encompassing residues 58-78. Because of consensus casein kinase II (CK II) sites in this peptide, we examined the in vitro phosphorylation of the purified recombinant VP1 protein by CK II. CK II phosphorylated VP1 on serine, and the resulting tryptic phosphopeptide eluted in a 30-31 min high performance liquid chromatography fraction corresponding to residues 58-78. The VP1 tryptic phosphopeptide also co-migrated in two-dimensional peptide analysis with one of the tryptic peptides obtained from VP1 isolated after in vivo 32P labeling of virus-infected cells. A site-directed mutant VP1 protein, Ser-66 to Ala, was phosphorylated poorly by CK II in vitro. As determined by electron microscopy, all of the mutant proteins were isolated in pentameric form similar to the wild-type protein, although the Ala-66 pentamers had a tendency to self-assemble in vitro into tubular as well as capsid-like structures. These findings identify Ser-66 as a site of VP1 phosphorylation in vitro, and suggest that VP1 may serve as a substrate for CK II in vivo.
Asunto(s)
Proteínas de la Cápside , Cápside/metabolismo , Poliomavirus/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3/virología , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Secuencia de Bases , Cápside/genética , Cápside/ultraestructura , Quinasa de la Caseína II , Cromatografía Líquida de Alta Presión , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Mapeo Peptídico , Fosforilación , Fosfoserina/análisis , Proteínas Recombinantes/aislamiento & purificación , Serina/metabolismo , Tripsina/metabolismoRESUMEN
We have examined the structure of S-layers isolated from Sulfolobus acidocaldarius using atomic force microscopy (AFM) and transmission electron microscopy (TEM). From the AFM images, we were able to directly observe individual dimers of the crystal, defects in the crystal structure, and twin boundaries. We have identified two types of boundaries, one defined by a mirror plane and the other by a glide plane. This work shows that twin boundaries are highly structured regions that are directly related to the organization of units within each crystal domain. Projection maps from TEM images have shown that there are significant differences in the final average maps has allowed us to relate high magnification views obtained by AFM to the relatively high resolution information obtained by electron microscopy and image processing.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Proteínas Bacterianas/aislamiento & purificación , Microscopía/métodos , Microscopía Electrónica/métodos , Sulfolobus acidocaldarius/químicaRESUMEN
In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions. At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy. However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I. When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion. Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value. A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly. A recombinant HPV type 11 L1 protein, which was purified after expression in Escherichia coli and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures. These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm.
Asunto(s)
Papillomavirus Bovino 1/fisiología , Proteínas de la Cápside , Cápside/metabolismo , Disulfuros , Proteínas Oncogénicas Virales/metabolismo , Ensamble de Virus , Animales , Papillomavirus Bovino 1/efectos de los fármacos , Papillomavirus Bovino 1/metabolismo , Cápside/efectos de los fármacos , Cápside/genética , Bovinos , Cisteína/genética , Cisteína/metabolismo , Glicina/genética , Glicina/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas Virales/genética , UltracentrifugaciónRESUMEN
The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to assemble further. In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells. The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein. The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.