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

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 β-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.

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.

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 Ca -actin were sensitive to the polymerization ion (K or Na ): ΔS = 39 or 36 (cal/mol K), ΔΗ= 3.

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.

Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and β-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.

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.

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.

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.

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.