Satoru Fujiwara

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study.

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.

Denaturation and aggregation of hen egg lysozyme in aqueous ethanol solution studied by dynamic light scattering.

We applied dynamic light scattering technique on the model system of hen egg lysozyme in salt-free aqueous ethanol solution to study the mechanism of denaturation and aggregation of protein. At low ethanol concentration [0-63% (v/v)], the fast relaxation mode was observed, which was caused by lysozyme molecules in the solution interacting with each other with strong repulsive electrostatic force. At 45 and 63% (v/v) ethanol, the slow relaxation mode was also observed, which showed translational diffusive nature, similar to that observed in salt-free polyelectrolyte solution. At 72 or 81% (v/v) ethanol, the slow mode disappeared, leaving only the fast mode. However, the mutual diffusion coefficients obtained from the fast mode at 72 and 81% (v/v) ethanol decreased by about one order of magnitude compared with those from the fast mode at 0-63% (v/v). The reported alcohol-induced conformational transformation of lysozyme molecules at >60% (v/v) ethanol from their native structure to an alpha-helix-rich structure might cause such drastic decrease in the mutual diffusion coefficients. At the highest ethanol concentration of 90% (v/v), the slow mode reappeared, and its relaxation rate was decreasing with elapsed time, which is possibly due to the growth of aggregates of lysozyme molecules. X-ray diffraction results suggested that the intermolecular beta-sheet formation caused the aggregation. Thus, our results indicated that the change in molecular structure of lysozyme closely relates to the diffusion of molecules and their aggregation.

Effects of Salt Concentration on Association of the Amyloid Protofilaments of Hen Egg White Lysozyme Studied by Time-resolved Neutron Scattering

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.

Repetition Rate Multiplication: RRM, an Advanced Measuring Method Planed for the Backscattering Instrument, DNA at the MLF, J-PARC

A Si-analyzer neutron backscattering spectrometer, DNA , in MLF is the first indirect geometry instrument which chooses the coupled moderator as a pulsed neutron source in addition with a high speed disc chopper as a pulse-shaping device, aiming for high energy resolution with high intensity. The pulse-shaping has the advantage of obtaining a variety of slit opening times. On the other hand, it has the disadvantage of limiting the wavelength band width measured within one pulse-shaped neutron beam. This disadvantage is solved by employing 4 slit discs on the pulse-shaping chopper and generating multiple pulse-shaped incident beams to realize efficient measuring, the so called Repetition Rate Multiplication: RRM technique. The automatic phasing procedure for the RRM technique is firstly discussed.

Modulation of the picosecond dynamics of troponin by the cardiomyopathy-causing mutation K247R of troponin T observed by quasielastic neutron scattering

Troponin (Tn), consisting of three subunits (TnC, TnI, and TnT), regulates cardiac muscle contraction in a Ca2+-dependent manner. Various point mutations of human cardiac Tn are known to cause familial hypertrophic cardiomyopathy due to aberration of the regulatory function. In this study, we investigated the effects of one of these mutations, K247R of TnT, on the picosecond dynamics of the Tn core domain (Tn-CD), consisting of TnC, TnI and TnT2 (183-288 residues of TnT), by carrying out the quasielastic neutron scattering measurements on the reconstituted Tn-CD containing either the wild-type TnT2 (wtTn-CD) or the mutant TnT2 (K247R-Tn-CD) in the absence and presence of Ca2+. It was found that Ca2+-binding to the wtTn-CD decreases the residence time of atomic motions in the Tn-CD with slight changes in amplitudes, suggesting that the regulatory function mainly requires modulation of frequency of atomic motions. On the other hand, the K247R-Tn-CD shows different dynamic behavior from that of the wtTn-CD both in the absence and presence of Ca2+. In particular, the K247R-Tn-CD exhibits a larger amplitude than the wtTn-CD in the presence of Ca2+, suggesting that the mutant can explore larger conformational space than the wild-type. This increased flexibility should be relevant to the functional aberration of this mutant.