Protein anatomy: C-tail region of human tau protein as a crucial structural element in Alzheimer's paired helical filament formation in vitro.

Tau is a microtubule-associated protein in mammalian brain. In Alzheimer's disease, this protein is present in the somatodendritic compartment of certain nerve cells, where it forms a portion of paired helical filament, the major constituent of the neurofibrillary tangle. For clarification of the mechanism of this formation, recombinant human tau and its fragments (N-terminal half, C-terminal half, and 4-repeats) expressed in Escherichia coli were prepared, eight peptide fragments (C-tails 1-8) of the C-tail region were synthesized, and the conformation and capacity for aggregation essential for filamentous structure formation in vitro were examined. Recombinant full-length tau, the N-terminal half, 4-repeats, and the C-terminal half did not form filamentous structures in aqueous solution after standing at 20 degrees C. Peptides corresponding to the C-tail region of tau, C-tail 5, C-tail 7, and C-tail 8, produced the paired filament or single straight filament in acidic solution. The rate of filament formation by each peptide was followed by circular dichroism, which showed the C-tails to have predominantly random coil structures immediately following dissolution in aqueous solution and be gradually converted to the beta-sheet structure. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, whose negative peak intensity at 218 nm in circular dichroism increased due to filamentous structure formation. This delay was eliminated by seeding supersaturated solution of preformed filaments. C-tails interacted with recombinant full-length tau to form definite single straight filament. The C-tail region of tau is thus shown indispensable to the formation of paired helical filament and nucleation to reduce the rate of paired helical filament formation in amyloidogenesis in vitro. These findings may provide some clarification of the pathogenesis of Alzheimer's disease.

Protein anatomy: spontaneous formation of filamentous helical structures from the N-terminal module of barnase.

This paper reports the conformation of the N-terminal module (24 amino acid residues) of barnase in aqueous solution. This module contains the first of three helices in the intact protein. Circular dichroism spectra showed the peptide fragment to have a predominantly random coil structure immediately following dissolution in aqueous solution and to be gradually converted to a helical structure at 5 degrees C. This was mediated by aggregation, and an electron micrograph indicated the aggregate to be comprised of filamentous helical structures. Scanning tunneling microscopy showed the filamentous structures to be made up of protofilamentous structures containing many disks apparently stacked on top of each other. A monomer of the peptide predominantly took on a random coil conformation in aqueous solution and the multimer, a stable helical structure. A local amino acid sequence would thus appear to determine the secondary structure corresponding to that in a native protein but stability to be governed by other factors such as tertiary interactions. Helical wheel representation indicated the peptide fragment to have the features of an amphiphilic helix. Hydrophobic burial may provide the driving force for producing a stable helical structure in aqueous solution.

Imaging of subunit complexes of thermophilic bacterium H(+)-ATPase with scanning tunneling microscopy.

Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H(+)-ATPase of a thermophilic bacterium. The measurement was carried out in air without conductive coating on the samples deposited on a highly oriented pyrolytic graphite (HOPG). The F1 subunit complex of the H(+)-ATPase, and an H(+)-ATPase whose F0 portion was embedded into liposomes prepared from soybean lecithin were imaged. Overall structural images of the subunit complex F1 were obtained: the structural dimensions of the STM images are in agreement with those deduced from conventional methods such as an transmission electron microscopy (TEM) and small-angle X-ray scattering (SAX) experimentation. Regarding the STM imaging of these samples, we discuss the advantages and disadvantages of the STM over those of conventional methods such as a TEM and SAX.

Dynamic light-scattering study on polymerization process of muscle actin.

Globular actin (G-actin) polymerizes into a fibrous form (F-actin) under physiological salt conditions. The polymerization process of muscle actin was studied by a dynamic light-scattering method. The intensity correlation functions G2(tau) of scattered light from a G-actin solution containing 2 mM Tris-HCl (pH 8.0) and 0.1 mM ATP were analyzed by a cumulant expansion method, and the translational diffusion coefficient was determined to be D = (8.07 +/- 0.10) X 10(-7) cm2/s at 20 degrees C. This D value gave a diameter of 5.3 nm for spherical G-actin including a hydration layer. Polymerization of 1-3 mg/ml G-actin in a solution containing 10 mM Tris-HCl (pH 8.0), 0.2 mM ATP and 60 mM KCl was followed by successive measurements of G2(tau) for a data accumulation period of 60-300 s/run. The time evolution of G2(tau) was analyzed by a least-squares fitting to the field correlation function of a multiexponential form g1(tau) = sigma iAi exp(-gamma i tau) with gamma 1 greater than gamma 2 greater than 3 greater than ..., and the static scattering intensity I(t) = mean value of I as a function of time t after initiation of polymerization was decomposed as I(t) = mean value of I sigma iAi. At the early stage of polymerization, a two-exponential fit gave results indicating that component 1 came from G-actin and component 2 from F-actin growing linearly with t. At the middle stage of polymerization, a three-exponential fit gave the results that component 1 came from G-actin and possibly its small oligomers, component 2 from polymers with a number-average length Ln of about 900 nm which was independent of t, and component 3 from 'ghosts' in dynamic light scattering in a semidilute regime. Component 3 was concluded to arise from restricted motions of polymers with lengths much longer than Ln in cages formed by polymers giving component 2, and a fragmentation-elongation process of F-actin was suggested to start at the middle stage of polymerization, resulting in the size redistribution of F-actin.

Asymmetry of fluctuation with respect to time reversal in steady states of biological systems.

The asymmetry of fluctuation with respect to time reversal which is expected in an energy-consuming steady state is discussed with special attention to biological systems. The necessary condition for asymmetry of fluctuation of an observed quantity is given. To show the usefulness of the experimental analysis of asymmetry of fluctuation, some calculations are carried out on two simple examples of three-state reactions. In one of them, the two-point time correlation function of the observed quantity has an oscillatory component, while in the other the function is nearly exponential, but in both cases, the fluctuation has a pronounced asymmetry. A method to estimate the degree of asymmetry of fluctuation is proposed, and the application of the present method to investigation of the molecular events in biological systems such as muscle is discussed.