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C Toyoshima

Publications and source records attributed to C Toyoshima.

At least 37 records · Page 2Linked to original sources
PubMed1996

Two-dimensional crystallization and cryo-electron microscopy of photosystem II.

Two dimensional (2D) crystals of photosystem II (PSII) were obtained from n-heptyl-beta-D-thioglucoside-solubilized monomers of spinach PSII complex by conventional detergent dialysis. The 2D crystals were either large cylindrical vesicles (1 to 2 micrometer by 4 to 6 micrometer as flattened vesicles) or large monolayer sheets (ca. 1 micrometer X 1 micrometer), both suitable for cryo-electron microscopy. Images of unstained crystals embedded in ice were recorded using low-dose microscopy and analyzed by digital image processing. Both types of crystals had the same unit cell size and the same packing arrangement of PSII particles. The plane group was p22(1)2(1) and the unit cell was rectangular with dimensions of 16.7 nm X 15.3 nm containing four monomers (two face-up and two face-down). SDS-PAGE and immunoblot analyses of the 2D crystal indicated that the constituent subunits of particles in the 2D were CP47, D1, D2, cytochrome b-559 and psbI protein. A projection map of 20 A resolution revealed that each monomer has asymmetrical shape with a length of 8.1 nm and a maximal width of 7.5 nm consisting of four areas of density. Two high-density areas with similar sizes were located close to each other to form a roughly rectangular core of 4.0 nm X 6.5 nm. From its size similarity to the size of the L/M heterodimer of the bacterial reaction center, this high density core area was tentatively assigned to the D1/D2 heterodimer. The remaining large and small areas were also tentatively assigned to CP47 and cytochrome b-559 plus psbI protein respectively.

Crystallization↗
PubMed1996

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum.

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.

Animals↗
PubMed1996

Three-dimensional structure of bovine cytochrome bc1 complex by electron cryomicroscopy and helical image reconstruction.

Cytochrome bc1 complex from bovine heart has been reconstituted into tubular crystals. The three-dimensional structure of the complex in lipid bilayer has been obtained at an effective resolution of 16 angstrom by electron cryomicroscopy and helical image reconstruction. The complex is in a dimeric form, in which the monomers are associated closely in extramembrane domains on both sides of the membrane. The large inner domain is distinctively hollow and the small outer domain consists of a flat mass and two bulbous extrusions. These domains are connected by two narrow transmembrane columns. Locations of the subunits and the redox centres in the model are proposed.

Animals↗
PubMed1996

Rapid analysis of four bilirubins in domestic animal sera using high-performance liquid chromatography.

A rapid method was developed to analyze delta-bilirubin (B delta), diconjugated bilirubin (DCB), monoconjugated bilirubin (MCB), and unconjugated bilirubin (Bu) by direct injection of sera using high-performance liquid chromatography (HPLC) with an internal-surface reversed-phase silica support (ISRP) column. Sharp bilirubin peaks were obtained using a simple mobile phase of acetonitrile: 0.5 M Tris-HCl buffer (20:80, v/v, pH 7.2). A variable-wavelength detector set at 450 nm, 0.01 absorbance unit full scale (AUFS), and a recorder set at 4 mm/min were used for detection. Peaks for B delta, DCB, MCB and Bu appeared at 4.4, 6.4, 9.2 and 14.5 min, respectively, in human serum from subject with obstructive jaundice which was used as a bilirubin standard throughout this experiment. The mean recovery rate after direct addition of Bu in swine serum was 91.9% and that of DCB was 95.9%. When sera from icteric cattle, pigs and horses were analyzed using the direct injection technique, four bilirubin peaks were obtained and there was reliable correlation between the sum of the bilirubin peak heights observed on HPLC and the total bilirubin value measured by a standard reference procedure.

Animals↗
PubMed1995

The structure of the R-type straight flagellar filament of Salmonella at 9 A resolution by electron cryomicroscopy.

The supercoiled forms of the flagellar filaments are thought to be constructed from a mixture of two distinct subunit conformations arranged in a regular manner. We analyzed the structure of one of the two straight flagellar filaments, each of which is built up with all its subunits in one of the two conformations. The filament we studied was isolated from the strain SJW1655 of Salmonella typhimurium and had a right-handed helical symmetry. With recent advancements in electron cryomicroscopy, such as a liquid helium temperature stage for frozen hydrated specimens and a stable field emission source, and also by averaging high resolution data with a proper correction of the contrast transfer function, the density distribution map of this straight flagellar filament was generated in far more detail than before by including data up to 9 A resolution. The structure shows a densely packed core region from about 15 to 55 A in radius, where a pair of concentric tubular features of high density is present without well-defined subunit boundaries, and an outer part from 55 to 115 A, where the subunits are mostly well separated from each other. The outer tube in the core region, from 35 to 55 A in radius, contains many rod-like features with near-axial orientation and closest lateral distances of around 10 A, which are most likely to represent the alpha-helical bundles that were predicted in our previous report. In the inner tube, from 15 to 30 A in radius, the rod-like features are less clear. Between the inner and outer tubes are the short spoke-like densities, which are radially tilted and are connecting the two tubes. The outer part, from 55 to 115 A, contains an axially elongated column density and a slewed projection with a narrow neck region. When compared with the other straight filament having left-handed helical symmetry, this outer part does not show any significant changes in orientation, suggesting that the switch in the subunit conformation and packing involved in the polymorphic transitions is quite subtle and only occurs within the core region. Reassignment of each structural domain to the amino acid sequence is suggested, based on the volume of each domain, which was determined rather precisely by a proper correction of the contrast transfer function for both amplitudes and phases.

Bacterial Proteins↗
PubMed1993

Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane.

The ATP-driven calcium pump (Ca(2+)-ATPase) is an integral membrane protein (M(r) 110K) which relaxes striated muscle by pumping calcium out of the cytoplasm into the sarcoplasmic reticulum against a large concentration gradient. Recent efforts have attempted to relate the sequence of Ca(2+)-ATPase to its structure and function. In particular, site-directed mutagenesis has identified critical amino-acid residues, and its predicted secondary structure, which includes ten transmembrane helices, has gained experimental support. But direct visualization of the molecule has so far been limited to the cytoplasmic domains at low resolution. We present here the three-dimensional structure of Ca(2+)-ATPase in the native sarcoplasmic reticulum membrane at 14 A resolution, determined by cryo-electron microscopy and helical image analysis. The structure shows an unexpected transmembrane organization, consisting of three distinct segments, one of which is highly inclined. These features can be related to earlier predictions of secondary structure.

Amino Acid Sequence↗
PubMed1993

Two-dimensional crystallization of catalase on a monolayer film of poly(1-benzyl-L-histidine) spread at the air/water interface.

Two-dimensional (2D) crystals of beef liver catalase were prepared by adsorption to a film of synthetic polypeptide, poly(1-benzyl-L-histidine) (PBLH), spread at the air/water interface. The crystallization experiments were carried out in the pH range of 4.8-6.4 for catalase solutions at low concentration (10 micrograms/ml). The pH-dependence suggested an electrostatic interaction in the binding of catalase to the PBLH film. At lower pH, small crystals were formed at a low binding rate, and at higher pH the binding was rapid and densely-packed 2D arrays with poor crystallinity were formed. To stimulate crystal growth, a thermal treatment was applied. One-shot heating of the interfacial catalase-PBLH film to 35-40 degrees C was remarkably effective to form larger 2D crystals. The structure of catalase 2D crystals has been analyzed by Fourier filtering of the transmission electron micrographs. The crystal form is a new one, containing four catalase molecules in the unit cell with lattice parameters of alpha = 187 A, b = 225 A and gamma = 92.8 degrees.

Animals↗
PubMed1992

Ligand-induced functions of the epidermal growth factor receptor require the positively charged region asymmetrically distributed across plasma membrane.

Many plasma membrane proteins, including the epidermal growth factor (EGF) receptor, possess basic regions on the cytoplasmic surface of the membrane. To examine the function of these positively charged regions, we constructed mutated EGF receptor genes lacking this region by substitution of the basic amino acid residues with 3 approximately 8 neutral Asn residues, or by their complete deletion. There was no significant difference in the affinities for EGF of the wild-type and mutant receptors which are produced in rodent fibroblasts through transfection. However, EGF-induced tyrosine phosphorylation of the receptor was strongly inhibited by removal of the 3 approximately 8 positively charged residues. On addition of EGF, cells expressing the mutant EGF receptors did not show morphological changes, whereas cells expressing the wild-type receptor did. These findings suggest that the positively charged regions of membrane proteins that are asymmetrically distributed on the cytoplasmic surface of the membrane may be required for the functions of membrane proteins in general.

3T3 Cells↗
PubMed1990

Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction.

Long tubular vesicles have been grown from isolated Torpedo postsynaptic membranes, in which the receptors are arranged helically on the vesicle surface. The structures of these tubes have been analyzed by cryoelectron microscopy of specimens embedded in thin films of ice, combined with helical image reconstruction. Complete data sets from tubes belonging to several helical families have been obtained to a resolution of 17 A in all directions. Confirming a preliminary study (Toyoshima, C., and N. Unwin. 1988. Nature (Lond.). 336:247-250), the central ion channel has an almost constant diameter throughout the molecule except for the portion extending through the hydrophobic part of the lipid bilayer, where the pore is too small to be resolved. However, the density on the pseudo fivefold axis running through the pore is consistently highest in the cytoplasmic half of the bilayer, suggesting the gate is located in that region. The path followed by each subunit has been identified throughout the length of the receptor. The two alpha subunits follow equivalent paths. All subunits have similar features which change in character at the same level relative to the membrane.

Animals↗
PubMed1989

Bidirectional movement of actin filaments along tracks of myosin heads.

It is well established that muscle contraction results from the relative sliding of actin and myosin filaments. Both filaments have definite polarities and well-ordered structures. Thick filaments, however, are not vital for supporting movement in vitro. Previously we have demonstrated that actin filaments can move continuously on myosin fragments (subfragment-1 or heavy meromyosin (HMM] that are bound to a nitrocellulose surface. Here we report that actin filaments can move in opposite directions on tracks of myosin heads formed when actin filaments decorated with HMM are placed on a nitrocellulose surface. The actin filaments always move forward, frequently changing the direction of the movement, but never move backward reversing the polarity of the movement. The direction of movement is therefore determined by the polarity of the actin filament. These results indicate that myosin heads have considerable flexibility.

Actins↗
PubMed1988

Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes.

The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time.

Animals↗
PubMed1988

Contrast transfer for frozen-hydrated specimens: determination from pairs of defocused images.

Electron imaging of frozen-hydrated biological molecules allows density maps to be obtained directly, without the need for fixatives or stains. The appearance of such maps may, however, be strongly influenced by the contrast transfer properties, which have not previously been evaluated by quantitative experiments. Here we determine the contribution due to amplitude contrast in a typical (approximately 300 A thick) frozen specimen, consisting of arrays of acetylcholine receptor, by comparing pairs of images recorded with different defocuses. We find that this specimen is imaged as a "weak-phase-weak-amplitude" object and that the contribution due to amplitude contrast is 7%.

Animals↗
PubMed1988

Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes.

Two conformational states of the nicotinic acetylcholine receptor have been investigated by cryoelectron microscopy of flattened vesicular crystals grown from Torpedo marmorata postsynaptic membranes. One was obtained from the vesicles without acetylcholine present, and is presumed to correspond to the native, or resting state; the other was obtained from the vesicles after exposure to 100 microM to 5 mM carbamylcholine (an acetylcholine analogue) and is presumed to correspond to a desensitized state. Both conformations were determined in three-dimensions to a resolution of 18 A, sufficient to reveal the configurations of the five subunits around the central ion channel over most of their length. The subunits of either structure have a similar appearance, consistent with their amino acid homology. They are each aligned almost parallel to the axis of the receptor, conferring a high degree of pentagonal symmetry to the bilayer portion and a contiguous region on the synaptic side. Their external surfaces form a pronounced ridge in the bilayer portion, which broadens toward the synaptic end. Comparison of features in the two three-dimensional maps reveals that carbamylcholine induces a quaternary rearrangement, involving predominantly the delta-subunit. The densities corresponding to this subunit are tilted by approximately 10 degrees tangential to the axis of the receptor over a large fraction of its length, and become misaligned relative to the densities corresponding to the other four subunits. The gamma-subunit is also affected, being displaced slightly away from the axis of the receptor. The alpha- and beta-subunits may be affected on a more localized scale. The overall changes are most pronounced in the synaptic region, where the ligand-binding site is located, and in the cytoplasmic region, which may be closer to the gate of the channel. The physiological process of desensitization appears to be associated with a structural transition in which the subunits switch to a less symmetrical configuration.

Animals↗
PubMed1988

Electron microscopic study on the location of 23 kDa and 50 kDa fragments in skeletal myosin head.

The functional activities of myosin head are located in a 95 kilodalton (kDa) heavy chain which can be divided into three fragments of 23 kDa, 50 kDa, and 20 kDa. ATP hydrolysis sites were suggested to be located in the 23 kDa and 50 kDa fragments, and actin binding sites were in the 50 kDa and 20 kDa fragments. In this study, we obtained electron microscopic images of the myosin molecule bound with antibodies directed to the 23 kDa and 50 kDa fragments. We determined that the antigenic sites for 23 kDa fragment are located at 140-180 A from the head-rod junction of myosin, and those for 50 kDa fragment at 160 A from the junction and at the tip of the head itself. The relationship between the spatial locations and the primary structures is discussed.

Animals↗
PubMed1988

Temperature-induced change of thick filament and location of the functional sites of myosin.

The combination of the small-angle X-ray camera to use the synchrotron radiation (National Lab. of High Energy Physics, Tsukuba) and a sensitive X-ray detecting system (FCR System, Fuji Medical System Inc.) using the imaging plate enabled us to record a small angle X-ray diagram of liver rabbit muscle within few minutes. Live relaxed rabbit muscle kept at 25 degrees C gave clear relaxed pattern, in which myosin layer line can be observed up to 13th order. When it was cooled down to 5 degrees C, it gave the small-angle X-ray pattern which looks like that obtained from contracting frog muscle, whereas cooled muscle produced no tension. The 8th meridional reflection almost vanished. The pattern is different from that obtained from rigor muscle: actin layer line at 72 A spacing can not be observed. The "stiffness" and (1,1) reflection on the equator increased. This indicates that more crossbridges are at the vicinity of the thin filaments without developing tension at low temperature. This might be related to the marked decrease in the rate of the step from M*ADP to M.ADP at low temperature. The position of the ATP binding site, SH1, actin binding site(s) was determined by three-dimensional image reconstruction method. Actin-binding site was determined by comparing the three-dimensional image of actin-tropomyosin-S1 and that of actin-tropomyosin-troponin-Ca. The position of ATP binding site and SH1 was determined by three-dimensional reconstruction of the complexes of actin-tropomyosin and S1 of which the ATP binding site or SH1 was labelled with avidin. It was found that SH1 locates near the actin-binding site at the same side of S1. The distance from head-rod junction to ATP binding site and SH1 was similar. But they locate at the different side of S1 and the distance between two sites is about 5 nm, which is consistent with that obtained by energy transfer method.

Animals↗
PubMed1985

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. IV. Reconstitution from minimal- and high-dose images of the actin-tropomyosin-myosin subfragment-1 complex.

Three-dimensional images of the actin-tropomyosin-myosin subfragment-1 (S1) complex were reconstituted from both minimal- and high-dose electron micrographs by using a conventional reconstruction technique. Higher resolution (1/15 A-1) than those of the previous reconstructions was attained. A multi-domain structure similar to that of the actin-S1 complex described in the previous paper (1) was observed and a ne diagram of the multi-domain structure of the actin-tropomyosin-S1 complex is presented. The shape of S1 molecules in the rigor complex was clearly resolved. In a view perpendicular to the filament axis, S1 had an axially bent profile; only the tail portion, which was thin but was not small in diameter, was steeply inclined. These features were more prominent in the model from minimal-dose images than that from high-dose images.

Actins↗
PubMed1985

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. V. Assignment of actin in the actin-tropomyosin-myosin subfragment-1 complex.

To assign the actin molecule in the three-dimensional image of the actin-tropomyosin-myosin subfragment-1 (actin-TM-S1) complex, the three-dimensional image of the actin-tropomyosin complex was correlated to that of actin-TM-S1. To assess the similarity of two structures in a quantitative manner, we used a normalized cross-correlation function ("similarity function"). The calculation of similarity indicated that domain A and domain B defined in (1, 2) correspond to actin-tropomyosin. This assignment indicates that one S1 molecule strongly interacts with only one actin molecule, but at least two regions of S1 contribute to the binding. Comparison of the reconstituted models of thin filaments with those of decorated thin filaments suggested a change in the shape of the actin molecule.

Actins↗