The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation.

In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.

Expression, Functional Characterization, and Preliminary Crystallization of the Cochaperone Prefoldin from the Thermophilic Fungus Chaetomium thermophilum.

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double ß-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin⁻chaperonin system.

Thermodynamic measurements of actin polymerization with various cation species.

We measured the critical concentration of actin polymerized with different polymerization ions and bound divalent cations at low temperatures and estimated thermodynamic parameters. The entropy and enthalpy changes of actin polymerization were 36-55 (cal/mol K) and 2-8 (kcal/mol), respectively, with some exceptions. Both the entropy and enthalpy changes of the polymerization of Ca2+ -actin were sensitive to the polymerization ion (K+ or Na+ ): ΔS = 39 or 36 (cal/mol K), ΔΗ= 3.9 or 2.7 (kcal/mol). The entropy and enthalpy changes (cal/mol K, kcal/mol) of Mg2+ -actin were also sensitive to the polymerization ion in the following order: Mg2+ (55, 7.6) > K+ (46, 5.3) > Na+ (38, 2.4). Those values largely decreased and became even negative in the presence of a high concentration (0.1 M) of K+ , which was likely caused by the charge screening effect of that ion.

Direct observation of the actin filament by tip-scan atomic force microscopy.

Actin filaments, the actin-myosin complex and the actin-tropomyosin complex were observed by a tip-scan atomic force microscope (AFM), which was recently developed by Olympus as the AFM part of a correlative microscope. This newly developed AFM uses cantilevers of similar size as stage-scan AFMs to improve substantially the spatial and temporal resolution. Such an approach has previously never been possible by a tip-scan system, in which a cantilever moves in the x, y and z directions. We evaluated the performance of this developed tip-scan AFM by observing the molecular structure of actin filaments and the actin-tropomyosin complex. In the image of the actin filament, the molecular interval of the actin subunits (∼5.5 nm) was clearly observed as stripes. From the shape of the stripes, the polarity of the actin filament was directly determined and the results were consistent with the polarity determined by myosin binding. In the image of the actin-tropomyosin complex, each tropomyosin molecule (∼2 nm in diameter) on the actin filament was directly observed without averaging images of different molecules. Each tropomyosin molecule on the actin filament has never been directly observed by AFM or electron microscopy. Thus, our developed tip-scan AFM offers significant potential in observing purified proteins and cellular structures at nanometer resolution. Current results represent an important step in the development of a new correlative microscope to observe nm-order structures at an acceptable frame rate (∼10 s/frame) by AFM at the position indicated by the fluorescent dye observed under a light microscope.

Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and ß-structure formation.

Members of the kinesin-13 sub-family, including KIF2C, depolymerize microtubules. The positive charge-rich 'neck' region extending from the N-terminus of the catalytic head is considered to be important in the depolymerization activity. Chemically synthesized peptides, covering the basic region (A182-E200), induced a sigmoidal increase in the turbidity of a microtubule suspension. The increase was suppressed by salt addition or by reduction of basicity by amino acid substitutions. Electron microscopic observations revealed ring structures surrounding the microtubules at high peptide concentrations. Using the peptide A182-D218, we also detected free thin straight filaments, probably protofilaments disintegrated from microtubules. Therefore, the neck region, even without the catalytic head domain, may induce lateral disintegration of microtubules. With microtubules lacking anion-rich C-termini as a result of subtilisin treatment, addition of the peptide induced only a moderate increase in turbidity, and rings and protofilaments were rarely detected, while aggregations, also thought to be caused by lateral disintegration, were often observed in electron micrographs. Thus, the C-termini are not crucial for the action of the peptides in lateral disintegration but contribute to structural stabilization of the protofilaments. Previous structural studies indicated that the neck region of KIF2C is flexible, but our IR analysis suggests that the cation-rich region (K190-A204) forms ß-structure in the presence of microtubules, which may be of significance with regard to the action of the neck region. Therefore, the neck region of KIF2C is sufficient to cause disintegration of microtubules into protofilaments, and this may contribute to the ability of KIF2C to cause depolymerization of microtubules.

Flexural rigidity of individual microtubules measured by a buckling force with optical traps.

We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 x 10(-24) Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 x 10(-24) Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules.

Evidence for a relationship between activity and the tetraprotomeric assembly of solubilized pig gastric H/K-ATPase.

Activity-oligomeric assembly relationships using octaethylene glycol dodecyl ether (C12E8) solubilized pig gastric H/K-ATPase (unmodified H/K-ATPase) or H/K-ATPase modified with Fluorescein 5'-isothiocyanate (FITC-H/K-ATPase) were examined. The amount of oligomeric species in FITC-H/K-ATPase, which retained little H/K-ATPase activity was estimated by a single-molecule detection technique using total internal reflection fluorescence microscopy. Solubilization of the FITC-H/K-ATPase reduced the potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity to around 5% of the level of the membrane-bound enzyme with the formation of 50% protomer and 40% diprotomer. The solubilization of unmodified H/K-ATPase also reduced both the K-pNPPase and H/K-ATPase activities to around 5%. However, solubilization with increasing concentrations of potassium acetate induced significant and similar increases in K-pNPPase activity (K0.5 = 35 mM) with an increase in the amount of the tetraprotomer of FITC-H/K-ATPase, and the K-pNPPase (K0.5 = 28 mM) and H/K-ATPase (K0.5 = 40 mM) activities of the unmodified H/K-ATPase. The correlation coefficient between the proportion of tetraprotomer and the proportion of the K-pNPPase activity for the same FITC-H/K-ATPase preparation was estimated to be 0.93. Similar coefficients were also obtained between the proportion of tetraprotomer in the FITC-H/K-ATPase and the proportion of K-pNPPase and H/K-ATPase activities in the unmodified H/K-ATPase, with value of 0.85 and 0.86, respectively. Such positive correlations were not obtained between these activities and other oligomeric species. These data, the first direct comparison of oligomeric assembly and enzyme activity both stabilized by K+ in C12E8-solubilized gastric H/K-ATPase, provide strong evidence that the catalytic unit of C12E8-solubilized gastric H/K-ATPase is a tetraprotomer.

Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase.

Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species in the membrane.

Partial specific volume and adiabatic compressibility of G-actin depend on the bound nucleotide.

We determined the partial specific volume and partial specific adiabatic compressibility of either ATP- or ADP-bound monomeric actin in the presence of Ca(2+) by measuring the density of and sound velocity in a monomeric actin solution at 18 degrees C. The partial specific volume of ATP-bound monomeric actin, equal to 0.744 cm(3)/g, which is exceptionally high among globular proteins, was reduced to 0.727 cm(3)/g when the tightly bound ATP was replaced with ADP. Associated with this, the adiabatic compressibility of ATP-bound monomeric actin, equal to 8.8 x 10(-12) cm(2)/dyne, decreased to 5.8 x 10(-12) cm(2)/dyne, which is a common value for globular proteins. These results suggested that an extraordinarily soft global conformation of ATP-bound monomeric actin is packed into a compact mass associated with the hydrolysis of bound ATP. When monomeric actin was limitedly proteolyzed at subdomain 2 with subtilisin, the nucleotide-dependent flexibility of the global conformation of monomeric actin was lost.