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Tetsuro Fujisawa

Publications and source records attributed to Tetsuro Fujisawa.

At least 19 recordsLinked to original sources
PubMed2006

pH-dependent formation of membranous cytoplasmic body-like structure of ganglioside G(M1)/bis(monoacylglycero)phosphate mixed membranes.

Membrane structures of the mixtures of ganglioside G(M1) and endosome specific lipid, bis (monoacylglycero) phosphate (BMP, also known as lysobisphosphatidic acid) were examined at various pH conditions by freeze-fracture electron microscopy and small-angle x-ray scattering. At pH 8.5-6.5, a G(M1)/BMP (1:1 mol/mol) mixture formed small vesicular aggregates, whereas the mixture formed closely packed lamellar structures under acidic conditions (pH 5.5, 4.6) with the lamellar repeat distance of 8.06 nm. Since BMP alone exhibits a diffuse lamellar structure at a broad range of pH values and G(M1) forms a micelle, the results indicate that both G(M1) and BMP are required to produce closely stacked multilamellar vesicles. These vesicles resemble membranous cytoplasmic bodies in cells derived from patients suffering from G(M1) gangliosidosis. Similar to G(M1) gangliosidosis, cholesterol was trapped in BMP vesicles in G(M1)- and in a low pH-dependent manner. Studies employing different gangliosides and a G(M1) analog suggest the importance of sugar chains and a sialic acid of G(M1) in the pH-dependent structural change of G(M1)/BMP membranes.

Biophysics↗
PubMed2006

Autoxidized phospholipids in hexane: nano-self-assemblies studied by synchrotron small-angle X-ray scattering.

Synchrotron small-angle X-ray scattering (SAXS) was used to analyze the structure of self-assembled autoxidized phospholipids in a very dilute solution of hexane. In addition, it was used to build a self-consistent model of the aggregates, taking into account their inner heterogeneities and polydispersity. The scattering intensity from a dilute mixture of different types of noninteracting components of the phospholipid system was represented as a linear combination of partial intensities from the components weighted by their volume fractions. Applying this approach the final model of the system was described as a mixture of polydisperse reverse micelles and aggregates with spherical and cylindrical shapes. Spherical aggregates were represented as hollow spheres with inner radius 0.7 nm (occupied by water or hexane) and outer radius 1.5 nm. Geometrical parameters of the aggregates did not change much during the oxidation process, while the ratio of reverse micelles and aggregates in solution varied. The amount of the reverse micelles increased from very low to about 80%, whereas the content of other aggregates constantly reduced. The analysis performed in this study helps one to better understand the processes of phospholipid oxidation, which may occur in biological membranes.

Hexanes↗
PubMed2006

Differential membrane packing of stereoisomers of bis(monoacylglycero)phosphate.

Bis(monoacylglycero)phosphate (BMP) reveals an unusual sn-1,sn-1' stereoconfiguration of glycerophosphate. We synthesized sn-(3-myristoyl-2-hydroxy)glycerol-1-phospho-sn-1'-(3'-myristoyl-2'-hydroxy)glycerol (1,1'-DMBMP) and characterized the thermotropic phase behavior and membrane structure, in comparison with those of the corresponding sn-3:sn-1' stereoisomer (3,1'-DMBMP), by means of differential scanning calorimetry (DSC), small- and wide-angle X-ray scattering (SAXS and WAXS, respectively), pressure-area (pi-A) isotherms, epifluorescence microscopy of monolayers, and molecular dynamics (MD) simulations. In DSC, these lipids exhibited weakly energetic broad peaks with an onset temperature of 9 degrees C for 1,1'-DMBMP and 18 degrees C for 3,1'-DMBMP. In addition, a highly cooperative, strongly energetic transition peak was observed at approximately 40 degrees C for 1,1'-DMBMP and approximately 42 degrees C for 3,1'-DMBMP. These results are supported by the observation that 1,1'-DMBMP exhibited a larger phase transition pressure (pi(c)) than 3,1'-DMBMP. Small- and wide-angle X-ray scattering measurements identified these small and large energetic transitions as a quasi-crystalline (L(c1))-quasi-crystalline with different tilt angle (L(c2)) phase transition and an L(c2)-L(alpha) main phase transition, respectively. X-ray measurements also revealed that these DMBMPs undergo an unbinding at the main phase transition temperature. The MD simulations estimated stronger hydrogen bonding formation in the 3,1'-DMBMP membrane than in 1,1'-DMBMP, supporting the experimental data.

Calorimetry, Differential Scanning↗
PubMed2006

The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering.

The structure of a histidine kinase (ThkA) complexed with a response regulator (TrrA) in the two-component regulatory system from hyperthermophile Thermotoga maritima was determined by a combination of X-ray crystallography at a resolution of 4.2 A and small-angle X-ray scattering (SAXS). The boundary of the three component domains (PAS-sensor, dimerization and catalytic domains) of ThkA and the bound TrrA molecule were unambiguously assigned in the electron density map at 4.2 A resolution. ThkA forms a dimer with crystallographic 2-fold symmetry and two monomeric TrrAs bind to the ThkA dimer. SAXS experiments also confirmed this association state in solution and specific binding between ThkA and TrrA (Kd=8.2x10(-11) M(-2)). The association interface between ThkA and TrrA contains the phosphotransfer His residue in the ThkA, indicative of an efficient receipt of the phosphoryl group. One Per-Arnt-Sim (PAS) domain does not interact with the other PAS domain, but with the catalytic domain of the same polypeptide chain and with one TrrA molecule. Observed inter-domain and inter-molecular interactions reveal a definite pathway of signal transduction in the kinase/regulator complex. In addition, we propose a responsible role of TrrA for the feedback regulation of sensing and/or kinase activities of ThkA.

Amino Acid Sequence↗
PubMed2006

D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol alters cellular cholesterol homeostasis by modulating the endosome lipid domains.

D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is a frequently used inhibitor of glycosphingolipid biosynthesis. However, some interesting characteristics of D-PDMP cannot be explained by the inhibition of glycolipid synthesis alone. In the present study, we showed that d-PDMP inhibits the activation of lysosomal acid lipase by late endosome/lysosome specific lipid, bis(monoacylglycero)phosphate (also called as lysobisphosphatidic acid), through alteration of membrane structure of the lipid. When added to cultured fibroblasts, D-PDMP inhibits the degradation of low-density lipoprotein (LDL) and thus accumulates both cholesterol ester and free cholesterol in late endosomes/lysosomes. This accumulation results in the inhibition of LDL-derived cholesterol esterification and the decrease of cell surface cholesterol. We showed that D-PDMP alters cellular cholesterol homeostasis in a glycosphingolipid-independent manner using L-PDMP, a stereoisomer of D-PDMP, which does not inhibit glycosphingolipid synthesis, and mutant melanoma cell which is defective in glycolipid synthesis. Altering cholesterol homeostasis by D-PDMP explains the unique characteristics of sensitizing multidrug resistant cells by this drug.

1-Deoxynojirimycin↗
PubMed2006

Time-resolved small-angle X-ray scattering investigation of the folding dynamics of heme oxygenase: implication of the scaling relationship for the submillisecond intermediates of protein folding.

Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (Rg) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method. The folding process of HO was found to contain a transient oligomerization; however, the conformation within 10 ms was demonstrated to be monomeric and to possess Rg of 26.1(+/-1.1) A. Together with the corresponding data for proteins with different chain lengths, the seven Rg values demonstrated the scaling relationship to chain length with a scaling exponent of 0.35+/-0.11, which is close to the theoretical value of 1/3 predicted for globules in solutions where monomer-monomer interactions are favored over monomer-solvent interactions (poor solvent). The finding indicated that the initial folding dynamics of proteins bears the signature of the coil-globule transition, and offers a clue to explain the folding mechanisms of proteins with different chain lengths.

Animals↗
PubMed2005

Specifically collapsed intermediate in the early stage of the folding of ribonuclease A.

Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 micros after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at approximately 22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations.

Anilino Naphthalenesulfonates↗
PubMed2005

X-ray microdiffraction and conventional diffraction from frozen-hydrated biological specimens.

A system for recording microdiffraction patterns from micrometer-sized quick-frozen hydrated biological specimens at the high-flux beamline BL40XU of SPring-8 is described. The optics consists of a pair of pinholes drilled into tantalum substratum, with a defining aperture of diameter 2 microm. The frozen specimens are placed in an in-vacuum cryochamber mounted on a three-axis goniometer, where the specimens are stably held at a liquid-nitrogen temperature ( approximately 74 K). A beam size of 1.5 microm (full width at half-maximum) is attained at the sample position. By using this system, diffraction patterns have been recorded from an isolated single myofibril (diameter approximately 3 microm) of an insect flight muscle in an area equivalent to a single sarcomere (length approximately 3 microm). The technique is potentially applicable to other micrometer-sized hydrated biological specimens, which are more susceptible to radiation damage than dry synthetic polymers or biopolymers. The quick-freezing of biological specimens has also been proven useful in reducing the specimen volume in the beam in conventional diffraction recordings.

Animals↗
PubMed2005

Specific collapse followed by slow hydrogen-bond formation of beta-sheet in the folding of single-chain monellin.

Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 micros after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of beta-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.

Circular Dichroism↗
PubMed2004

Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering.

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.

Fluorescence Resonance Energy Transfer↗
PubMed2004

Radiation damage to a protein solution, detected by synchrotron X-ray small-angle scattering: dose-related considerations and suppression by cryoprotectants.

In small-angle X-ray scattering experiments at high-brilliant synchrotron sources, protein aggregation results from radiation damage. The radiation-induced aggregation of lysozyme in solution was qualitatively evaluated based on forward scattering and radii of gyration. The scattering did not change below 400 Gy and increased exponentially above this dose. The aggregation is only seen beyond the critical dose rate, and the 'dilution effect' known in radiology was also observed. Mass spectroscopy of the lysozyme solution exposed to a monochromatic X-ray beam did not show any cleavage of the polypeptide chain. Small-angle X-ray scattering patterns suggested that the radiation-induced aggregation should be a non-specific association of intact lysozyme, without substantial alterations of the folding topologies. It was found that the addition of small amounts of cryoprotectants, such as glycerol, ethylene glycol and sucrose, effectively reduced the radiation damage. Glycerol and ethylene glycol were identically effective in the 100 mM concentration range. A similar effective concentration was observed for sucrose. The damage reduction by the cryoprotectants was mainly ascribed to changes in the protein-protein interactions, and rarely to decreases in the diffusion rates of activated species.

Cryoprotective Agents↗
PubMed2004

Activation mechanisms of transcriptional regulator CooA revealed by small-angle X-ray scattering.

CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration Rg and the second virial coefficient (A2) were 25.3(+/-0.5)A and -0.39(+/-0.25) x 10(-4)ml mol g(-2), respectively. CO binding caused a slight increase in Rg (by 0.5A) and a marked decrease in A2 (by 5.09 x 10(-4)ml mol g(-2)). The observed decrease in A2 points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of Rg rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental Rg and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8 degrees around the axis of 2-fold symmetry.

Amino Acids↗
PubMed2004

Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.

The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.

Animals↗
PubMed2004

Intensity of X-ray reflections from skeletal muscle thin filaments partially occupied with myosin heads: effect of cooperative binding.

For quantitative analysis of contractile proteins of muscle by means of X-ray diffraction, it is important to know how the intensities of individual reflections are related to the number of diffracting objects, i.e., the amount of constituent contractile protein in the muscle cell. Here we diffused various amounts of exogenous myosin subfragment-1 (S1) into overstretched skinned skeletal muscle fibers, either in the presence or absence of Ca2+ , and derived the relationship between the S1 content and the intensities of reflections arising from the S1. In theory, the intensities should be proportional to the square of the S1 content (square law). However, the intensity-content relation deviated systematically from the square law as the S1 content was lowered, and it was better described as a linear function at the lower end of the S1 contents (<20% of saturation level). Model calculations show that the way of deviation is explained by the cooperative manner of S1 binding to the regulated thin filament.

Actins↗
PubMed2003

Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components.

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I' domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2.E3BP was thought to be 60 E2 plus approximately 12 E3BP. We have prepared homogenous human components. E2 and E2.E3BP have s(20,w) values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2.E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2.E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound approximately 60 E1 and maximally sedimented 64.4 +/- 1.5 S faster than E2, whereas E1-saturated E2.E3BP maximally sedimented 49.5 +/- 1.4 S faster than E2.E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (approximately 12) were bound by E2.E3BP than by E2. The findings of a smaller E2.E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I' domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248.E3BP12 mass, which is consistent with this model.

Dihydrolipoamide Dehydrogenase↗
PubMed2003

Expansion of polyglutamine induces the formation of quasi-aggregate in the early stage of protein fibrillization.

We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 degrees C and that its formation was completed in approximately 3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to approximately 90 monomers and a bowl-like structure with long and short axes of 400 and 190 A, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 A-1 corresponding to the 4.8-A spacing of beta-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that beta-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried beta-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine.

Animals↗