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Y Fujiyoshi

Publications and source records attributed to Y Fujiyoshi.

At least 37 records · Page 2Linked to original sources
PubMed1999

The projection structure of the membrane protein microsomal glutathione transferase at 3 A resolution as determined from two-dimensional hexagonal crystals.

The formation of two-dimensional crystals of the membrane-bound enzyme microsomal glutathione transferase is sensitive to fractional changes in the lipid-to-protein ratio. Variation of this parameter results in crystal polymorphism. The projection structure of a p6 crystal form of the enzyme has been determined by the use of electron crystallography. The unit cell at 3 A resolution is comprised of two trimers. The hexagonal p6 and the orthorhombic p21212 crystal types have common elements in the packing arrangement which imply dominant crystal contacts. An overall structural similarity between the protein molecules in the two crystal forms is suggested by the projection maps. Furthermore, a comparison of the p6 and p21212 projection maps identifies additional corresponding protein densities which could not be assigned to the microsomal glutathione transferase trimer previously. Surprisingly, an ambiguity of the rotational orientation was found for trimers interspersed at certain positions within the crystal lattice.

Animals↗
PubMed1999

Interactions of endothelin receptor subtypes A and B with Gi, Go, and Gq in reconstituted phospholipid vesicles.

To understand the biochemical basis for the functional divergence of the human endothelin receptor subtypes A (ETAR) and B (ETBR), they were expressed, purified from insect Sf9 cells, and reconstituted into phospholipid vesicles with the Go, Gq, and Gi proteins. For each G protein, a unique pattern of reactivity was observed with the different receptor subtypes. Both ETAR and ETBR activated Go to a similar maximal extent, and both subtypes activated Gq with similar EC50 values; however, the ETAR displayed a 2-3-fold higher maximal extent of activation. In contrast, both subtypes activated Gi to a similar maximal extent, but the ETAR displayed a 4-fold higher EC50 value as compared to the ETBR. To test whether these coupling specificities are influenced by C-terminal palmitoylation of the receptor, we mutated a cluster of cysteine residues near the end of the seventh transmembrane helix in both receptors. While the cysteine mutations in the ETBR resulted in a partially palmitoylated receptor, the replacement of these cysteine residues in the ETAR yielded a mostly palmitoyl-deficient receptor and had no effect on Go activation, but caused a reduction in the extents of Gi and Gq stimulation. Together, these studies provide important insights into the specificity of G protein coupling in the endothelin receptors. The ability to discriminate between the different G proteins under various physiological conditions may be a key element in the selection of distinct signal transduction pathways by the two receptor subtypes.

Amino Acid Sequence↗
PubMed1999

The structure of bacteriorhodopsin at 3.0 A resolution based on electron crystallography: implication of the charge distribution.

Electron crystallography has the potential to visualise the charge status of atoms. This is due to the significantly different scattering factors of neutral and ionised atoms for electrons in the low-resolution range (typically less than 5 A). In previous work, we observed two different types of densities around acidic residues in the experimental (|Fo|) map of bacteriorhodopsin (bR), a light-driven proton pump. We suggested that these might reflect different states of the acidic residues; namely, the protonated (neutral) and the deprotonated (negatively charged) state. To evaluate the observed charge more quantitatively, we refined the atomic model for bR and eight surrounding lipids using our electron crystallographic data set between 8.0 and 3.0 A resolution, where the charge effect is small. The refined model yielded an R-factor of 23.7% and a free R-factor of 33.0%. To evaluate the effect of charges on the density map, we calculated a difference (|Fo|-|Fc|) map including data of a resolution lower than 8.0 A resolution, where the charge effect is significant. We found strong peaks in the difference map mainly in the backbone region of the transmembrane helices. We interpreted these peaks to come from the polarisation of the polar groups in the main chain of the alpha-helices and we examined this by assuming a partial charge of 0.5 for the peptide carbonyl groups. The resulting R and free R-factors dropped from 0.250 and 0.341 to 0.246 and 0.336, respectively. Furthermore, we also observed some strong peaks around some side-chains, which could be assigned to positively charged atoms. Thus, we could show that Asp36 and Asp102 are likely to interact with cations nearby. In addition, peaks found around the acidic residues Glu74, Glu194 and Glu212 have different features and might represent positive charges on polarised water molecules or hydroxonium ions.

Amino Acid Sequence↗
PubMed1999

Influence of various nucleotides on the in situ crystallization of Ca2+-ATPase.

A reproducible in situ crystallization of the Ca2+-ATPase in isolated sarcoplasmic reticulum (SR) membranes was studied. The addition of various nucleotides to the washing buffer allowed the formation of tubular crystals, which is induced by vanadate. SR membranes washed with nucleotide-free buffer could not form tubular crystals upon subsequent incubation with vanadate.

Adenosine Triphosphate↗
PubMed1999

Trehalose embedding technique for high-resolution electron crystallography: application to structural study on bacteriorhodopsin.

Trehalose was compared with glucose in its use as an embedding medium for the preparation of two-dimensional crystals for electron crystallographic structure analysis. Purple membranes (two-dimensional crystals of bacteriorhodopsin) embedded in either sugar were studied in a 'partially hydrated' state as well as after 'drying' of the specimen. Diffraction patterns of the four conditions, all showing spots to a resolution better than 3.0 A, were analysed and merged. In the case of glucose embedded specimens, the merging R-factor obtained from the diffraction patterns of dried specimens was significantly better than the one obtained from partially hydrated specimens. In the case of trehalose embedding, the merging R-factor for partially hydrated specimens was significantly better than the dried one. The trehalose embedded, partially hydrated specimens gave the best merging R-factor in all four cases.

Bacteriorhodopsins↗
PubMed1998

Role of the outermost subdomain of Salmonella flagellin in the filament structure revealed by electron cryomicroscopy.

A mutant strain of Salmonella typhimurium, SJW46, has flagellar filaments supercoiled in the same form as the wild-type strain, SJW1103, and swims normally. However, its flagellar filaments are mechanically unstable and show anomalous behaviors of polymorphism. Flagellin from SJW46 has a large central deletion from Ala204 to Lys292 of SJW1103 flagellin, which has been thought to be located in the outer surface of the filament. Since the filament structure is determined by intersubunit interactions of the terminal regions in the densely packed core of the filament, no serious involvement of the deleted portion was expected in the filament stability and polymorphism. In order to locate the deleted portion and to understand the underlying mechanism of these anomalous characteristics, we carried out structure analysis of the L-type straight filament reconstituted from a mutant flagellin of SJW46 (SJW46S) and compared the structure with that of the SJW1660 filament, which is also the L-type but composed of flagellin with no deletion. The deleted portion was identified as the outermost subdomain, and the structure in the core region showed no appreciable differences. The structure revealed the previously identified folding of flagellin in further detail, and the significance of intersubunit interactions between outer domains, which are present in the SJW1660 filament but absent in the SJW46 filament. This suggests that these contacts have a significant contribution to the filament stability and polymorphic behavior, despite the fact that the contacting surface area occupies only a minor portion of the whole intersubunit interactions.

Caulobacter crescentus↗
PubMed1998

Expression, purification, and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide. large-scale purification of a functionally active G protein-coupled receptor produced in Sf9 insect cells.

Human pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was expressed in Sf9 insect cells and Chinese hamster ovary (CHO) cells. The recombinant receptor in Sf9 cell membranes had low affinity for 125I-PACAP27 (Kd = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTPgammaS), whereas the receptor in CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPgammaS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (Kd = 20-40 pM) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (Bmax = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (Kd = 17.3 pM) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (Mr = 48,000) was smaller than that of the receptor from CHO cells (Mr = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (Kd = 182.6 pM) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (Kd = 40.2 pM) and was GTPgammaS sensitive. [35S]GTPgammaS binding to the reconstituted Gs protein was enhanced by PACAP27 and PACAP38 (EC50 = 42.5 and 9.4 pM, respectively) but not by antagonist PACAP(6-38), indicating that the purified receptor was functionally active.

Animals↗
PubMed1998

Projection map of the reaction center-light harvesting 1 complex from Rhodopseudomonas viridis at 10 A resolution.

The photosynthetic reaction center-light harvesting 1 complex from Rhodopseudomonas viridis was purified and reconstituted into two-dimensional crystals. The single-layered crystalline sheets with lattice parameters a=b=133.3 A and gamma=120 degrees were investigated by electron cryo-microscopy and the projection map at 10 A resolution was calculated. The opening diameter of the light-harvesting ring of 72 A is sufficient to allow slight movement of the reaction center within the ring. Based on characteristic features observed in the projection map, the mechanism of energy transfer from the light-harvesting 1 complex to the reaction center was discussed.

Bacterial Proteins↗
PubMed1998

The structural study of membrane proteins by electron crystallography.

A high-resolution electron cryo-microscope equipped with a top-entry specimen stage has been refined by modifying a previously described superfluid helium stage. Instruments equipped with such a cryo-stage achieve a resolution of better than 2.0 A and have proved extremely powerful in the high-resolution structure analysis of membrane proteins. Improvement of the electron microscopic system in combination with improved specimen preparation techniques allowed the structure of bR to be analyzed to a resolution of 3.0 A. The 3D structure of bR, especially the surface features, revealed the structural basis for the efficient guidance of protons to the entrance of the transmembrane channel. Based on the characteristic difference of the atomic scattering factors for electrons of ionized atoms versus neutral atoms as well as the data analysis, charged and uncharged amino acid residues could be discriminated. Thus, electron crystallography is providing us with new and exciting insights into the structure of membrane proteins because it not only enables us to determine the structure of a membrane protein, but allows us to study its interaction with the surrounding lipid molecules and to determine its ionization state.

Animals↗
PubMed1997

Surface of bacteriorhodopsin revealed by high-resolution electron crystallography.

Bacteriorhodopsin is a transmembrane protein that uses light energy, absorbed by its chromophore retinal, to pump protons from the cytoplasm of bacteria such as Halobacterium salinarium into the extracellular space. It is made up of seven alpha-helices, and in the bacterium forms natural, two-dimensional crystals called purple membranes. We have analysed these crystals by electron cryo-microscopy to obtain images of bacteriorhodopsin at 3.0 A resolution. The structure covers nearly all 248 amino acids, including loops outside the membrane, and reveals the distribution of charged residues on both sides of the membrane surface. In addition, analysis of the electron-potential map produced by this method allows the determination of the charge status of these residues. On the extracellular side, four glutamate residues surround the entrance to the proton channel, whereas on the cytoplasmic side, four aspartic acids occur in a plane at the boundary of the hydrophobic-hydrophilic interface. The negative charges produced by these aspartate residues is encircled by areas of positive charge that may facilitate accumulation and lateral movement of protons on this surface.

Bacteriorhodopsins↗
PubMed1997

The 3.0 A projection structure of microsomal glutathione transferase as determined by electron crystallography of p 21212 two-dimensional crystals.

Two-dimensional crystals of rat microsomal glutathione transferase were grown during dialysis of detergent-solubilized enzyme after addition of a small amount of phospholipid. The crystals had two-sided plane group symmetry p21212 with a calibrated unit cell size of a=91.90 A, b=90.83 A. Electron diffraction patterns were recorded showing significant reflections extending to 3.0 A. A combination of these structure factor amplitudes with phases from high-resolution images following image processing was used to calculate a projection map of the protein. The asymmetric unit of the structure consists of three microsomal glutathione transferase molecules. The local 3-fold axis at the center of the trimer is delineated by six parallel alpha-helices, two from each monomer. The two helices differ significantly in their respective projection structure. The inner helical core of the trimer is partly surrounded by elongated domains with extensions towards the helices and which contain resolved density maxima at a spacing of 4 to 5 A. A well-defined strong peak is localized close to the elongated domain and at a distance of about 9.5 A from two of the inner helices.

Crystallization↗
PubMed1997

Characterization of human endothelin B receptor and mutant receptors expressed in insect cells.

Endothelin type-B receptor (ET(B)R) forms a stable complex with its ligand, endothelin-1. To facilitate biochemical and biophysical studies of human ET(B)R, several ET(B)R mutants carrying a hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells and were purified by a combination of biotinylated endothelin-1-ligand-affinity and nickel-affinity chromatographies. The ligand-free receptor was purified by dissociating the ligand x receptor complex with 2 M NaSCN, whereas the ligand-bound ET(B)R was purified by the use of thiol-sensitive biotinylated endothelin-1. While the wild-type ET(B)R was expressed at about 100 pmol 125I-endothelin-1-binding activity/mg membrane protein, the deletion of 36 residues from the N-terminus reduced the expressed activity to about 30%. On the other hand, the lack of glycosylation and the replacement of 2-9 residues in the N-terminal tail resulted in a 20-40% reduction in the expressed activity. Among the mutant proteins, [H57-H62, G63-G65]ET(B)R, carrying six His residues in the N-terminal tail, was studied extensively because it was purified most effectively. Ligand-free [H57-H62, G63-G65]ET(B)R, purified in digitonin, retained full ligand-binding activity, while other detergents led to partial denaturation of the receptor after solubilization or after elution with NaSCN. On the other hand, ligand-bound [H57-H62, G63-G65]ET(B)R could be purified in various detergents, such as n-octyl-beta-D-glucopyranoside or n-decyl-beta-D-maltopyranoside. Ligand-free [H57-H62, G63-G65]ET(B)R reconstituted in phospholipid vesicles stimulated the binding of guanosine 5'-3-O-(thio)triphosphate by Gq in the presence of endothelin-1. Ligand-bound [H57-H62, G63-G65]ET(B)R showed similar catalytic activity in nucleotide exchange by Gq. These results indicate that the ligand x receptor complex in a detergent-micellar solution retained the biologically active structure, and that the presence of ligand, endothelin-1, in the receptor molecule reinforces the stable assembly of a helical bundle and therefore the active structure.

Amino Acid Sequence↗
PubMed1997

The three-dimensional structure of aquaporin-1.

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.

Animals↗
PubMed1997

Central projection of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive trigeminal primary neurons in the rat.

Substance P (SP) is implicated in transmission of primary afferent nociceptive signals. In primary neurons, SP is colocalized with calcitonin gene-related peptide (CGRP), which is another neuropeptide marker for small to medium primary neurons. CGRP coreleased with SP augments the postsynaptic effect of SP and thereby modulates the nociceptive transmission. This study demonstrates the distribution of CGRP-like immunoreactivity (-ir) and SP-ir in the lower brainstem of normal rats and after trigeminal rhizotomy or tractotomy at the level of subnucleus interpolaris (Vi). By comparing the results obtained from normal and deafferented rats, we analyzed the central projection of trigeminal primary nociceptors. The CGRP-immunoreactive (-ir) trigeminal primaries projected to the entire rostrocaudal extent of the spinal trigeminal nucleus, the principal nucleus (PrV), the paratrigeminal nucleus (paraV), and the lateral subnucleus of solitary tract nucleus (STN) on the ipsilateral side. The trigeminal primaries projecting to the spinal trigeminal nucleus, paraV and STN also contained SP-ir. The ipsilateral trigeminal primaries were the exclusive source of CGRP-ir terminals in the PrV, the Vi and the dorsomedial nucleus within the subnucleus oralis (Vo). The medullary dorsal horn (MDH) and the lateral edge of Vo received convergent CGRP-ir projection from the ipsilateral trigeminal primaries and other neurons. The glossopharyngeal and vagal primaries are candidates for the source of CGRP-ir projection to the Vo and the MDH, while the dorsal root axons supply the MDH with CGRP-ir terminals. In addition, contralateral primary neurons crossing the midline appear to contain CGRP and to terminate in the MDH.

Animals↗
PubMed1997

Trigeminal primary projection to the rat brain stem sensory trigeminal nuclear complex and surrounding structures revealed by anterograde transport of cholera toxin B subunit-conjugated and Bandeiraea simplicifolia isolectin B4-conjugated horseradish peroxidase.

Trigeminal primary afferent neurons were labeled by injecting the rat trigeminal ganglion with either wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), cholera toxin B subunit (B)-HRP or Bandeiraea simplicifolia isolectin B4 (IB4)-HRP. B-HRP stained medium to large cells (> 600 microm2), while IB4-HRP mostly small cells (< 400 microm2). WGA-HRP labeled trigeminal ganglion neurons of all sizes. Cell bodies in the mesencephalic trigeminal tract nucleus were labeled with WGA-HRP and B-HRP but not IB4-HRP. B-HRP revealed dense projection to the entire brain stem sensory trigeminal nuclear complex (BSTC) except for lamina II of the medullary dorsal horn (MDH). Some contralateral projection was also seen in the caudal part of MDH. Non-trigeminal nuclei receiving B-HRP-labeled terminals included the paratrigeminal nucleus (paraV), solitary tract nucleus, supratrigeminal nucleus, Probst's nucleus and median accessory nucleus. Following IB4-HRP application, terminal label was found in more restricted regions within the BSTC. Modest terminal label was seen in the dorsal part of principal sensory nucleus and at the medial edge of subnucleus interpolaris, while relatively dense terminal fields were seen in the dorsal half of subnucleus oralis. The MDH laminae I and II contained dense terminal label. Non-trigeminal nuclei were almost devoid of the IB4-HRP-labeled terminals excepting the paraV that contained dense terminal label. The terminal areas revealed with WGA-HRP coincided with B-HRP-labeled and IB4-HRP-labeled areas combined.

Animals↗
PubMed1997

Details of the arrangement of the outer capsid of rice dwarf phytoreovirus, as visualized by two-dimensional crystallography.

Two-dimensional crystals were obtained from purified P8, an outer capsid protein of rice dwarf phytoreovirus. A filtered image of the two-dimensional crystal, in combination with the results of biochemical analysis, revealed the unit formation of the capsid protein, a capsomere structure, which appeared to be an approximately equilateral triangle with sides of approximately 6 nm and which was composed of a trimer of P8 protein. Details of the arrangements of the outer capsid of the virus are described.

Capsid↗