Shinpei Tanaka

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.

Protein crystallization induced by polyethylene glycol: A model study using apoferritin

The phase behavior of apoferritin solutions induced by the addition of polyethylene glycol (PEG) was studied. The interaction between apoferritin molecules was determined by dynamic light scattering. The comparison of the experiments with the theoretical calculations showed that the addition of NaCl to the protein solution only screened the electrostatic repulsion and did not induce attraction. By the addition of PEG, on the other hand, significant attraction was induced and three types of precipitation (crystals, liquid domains, and random aggregates) appeared depending on the concentration of PEG and on its molecular weight. The strength of the attraction could be explained by the depletion mechanism, although there was slight discrepancy between the simple theory and the experiments. Superiority of PEG is thus demonstrated since the depletion mechanism does not depend on specific nature of proteins. From the phase diagram, we suggest that the control of the concentration and molecular weight of PEG are...

Size and number density of precrystalline aggregates in lysozyme crystallization process

Using dynamic light scattering, we investigated supersaturated aqueous solutions of hen egg white lysozyme. We could observe the formation of aggregates only in solutions, from which crystals grew within a few days. The aggregates were grouped into smaller “units” and larger “clusters.” The units consisted of a few molecules, whereas the clusters grew from about 100 nm to 1 μm. At the beginning of aggregation, the number density of the units decreased, while that of the clusters increased. At this stage, unit-cluster aggregation proceeded. At the next stage, the number density of the units became constant, while that of the clusters began to decrease, which means that the units stopped aggregating and cluster-cluster aggregation started. The aggregation mechanism for the clusters fit well with the diffusion limited cluster aggregation model, but this model alone could not explain that the aggregates separated into two groups, corresponding to units and clusters, and that the units stopped aggregating duri...

Flurbiprofen ameliorated obesity by attenuating leptin resistance induced by endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins, is involved in the development of obesity. We demonstrated that flurbiprofen, a nonsteroidal anti‐inflammatory drug (NSAID), exhibited chaperone activity, which reduced protein aggregation and alleviated ER stress‐induced leptin resistance, characterized by insensitivity to the actions of the anti‐obesity hormone leptin. This result was further supported by flurbiprofen attenuating high‐fat diet‐induced obesity in mice. The other NSAIDs tested did not exhibit such effects, which suggested that this anti‐obesity action is mediated independent of NSAIDs. Using ferriteglycidyl methacrylate beads, we identified aldehyde dehydrogenase as the target of flurbiprofen, but not of the other NSAIDs. These results suggest that flurbiprofen may have unique pharmacological properties that reduce the accumulation of unfolded proteins and may represent a new class of drug for the fundamental treatment of obesity.

Kinetic study on the early stage of the crystallization process of two forms of lysozyme crystals by photon correlation spectroscopy

By using photon correlation spectroscopy, we have investigated the kinetics of the growth of clusters in two kinds of supersaturated lysozyme solutions at pH 4.6 and temperature 35°C from which orthorhombic rectangular crystals and needle-like crystals appear. Although the two kinds of crystals have quite different final morphologies, the increase of the cluster size in the early stage of the crystallization process can be commonly explained by a diffusion-limited aggregation model, which suggests that the clusters of random aggregates are formed in the early stage irrespective of the crystals systems or habits. We suggest that the needle-like crystals may be what these initial clusters grow to be.

Rationalization of Membrane Protein Crystallization with Polyethylene Glycol Using a Simple Depletion Model

Based on the importance of crystallizing membrane proteins in a rational way, cytochrome bc(1) complex (BC1) was crystallized using polyethylene glycol (PEG) as a sole crystallization agent. Interaction between protein-detergent complexes of BC1 was estimated by dynamic light scattering, and was compared with the numerical calculation using the Derjaguin-Landau-Verwey-Overbeek potential plus a depletion potential, without considering specific surface properties of the protein-detergent complexes. The experiments and calculation were found to be consistent and we obtained a relation between PEG molecular weight M and the range of depletion zone delta as delta approximately M(0.48+/-0.02). The stability of liquid phase of BC1 solutions was controlled by a ratio of (the range of depletion zone)/(the radius of a BC1 particle), which was consistent with recent theoretical predictions. The crystallization was most successful under a condition where the stability of the liquid phase changed from stable to unstable. The PEG molecular weight that fulfilled this condition coincided with the one used empirically to crystallize BC1 in the past by a number of groups. These results are compared to the fact that membrane proteins were often successfully crystallized close to the detergent cloud point.

Structural transitions of the mono-olein bicontinuous cubic phase induced by inclusion of protein lysozyme solutions

Inclusion of protein lysozyme molecules in the lipidic mono-olein cubic phase induces a transition from a Pn3m structure to an Im3m one. The small-angle x-ray scattering method with high-intensity synchrotron radiation enabled us to follow closely the transition depending on the conditions of lysozyme solutions. We show that concentrated lysozyme solutions induced the appearance of the Im3m structure coexisting with the Pn3m structure. From the relation between the lattice parameters of these two structures it is shown that they are related by the Bonnet transformation of the underlying triply periodic minimal surfaces. We found that the transition also occurred at lower lysozyme concentration when NaCl induced an attraction between lysozyme molecules. The origin of the transition was considered as a frustration in the cubic phase where lysozyme molecules were highly confined. A simple estimation of the frustration was given, which took into account the translational entropy of lysozyme molecules. At the highest concentration of lysozyme and NaCl the Im3m structure was found to disappear and left only the Pn3m structure. This was probably either due to the crystallization or phase separation of lysozyme solutions ongoing microscopically, which absorbed lysozyme molecules from channels of the cubic phase and thus removed the frustration.

The effect of amphiphilic additives on the growth and morphology of Aspergillus niger acid proteinase A crystals

Abstract The effect of amphiphilic additives, dimethyl sulfoxide (DMSO) and alkane diols (HO(CH 2 ) n OH , n=2 – 6) , on the crystallization of Aspergillus niger acid proteinase A was investigated by optical and confocal laser microscopy. As DMSO concentration was increased, while keeping ammonium sulfate concentration constant, the crystal clusters became larger and their number decreased. The crystal cluster morphology changed from thin needles to rod-like. The amphiphilic nature of DMSO molecules seemed to suppress unfavorable hydrophobic interaction between protein molecules in the solution and in the crystal. To control the amphiphilic nature of additives, an alkane diol series of n =2–6 was examined in addition to DMSO. Single crystals were obtained at n =4 and 5, while only crystal clusters were obtained at n =2 and 3 and no crystals appeared at n =6. These results suggest that the balance of hydrophobicity and hydrophilicity of additives is important in protein crystallization.

pH-dependent oligomerization of BPTI in undersaturated and supersaturated solutions studied by dynamic light scattering

Bovine pancreatic trypsin inhibitor (BPTI), a small protein molecule, is reported to form several types of oligomers in solution including a decamer. We investigate in this work the relation between the oligomerization and crystallization. The oligomerization and interaction between BPTI molecules in NaCl and NaH2PO4/K2HPO4 solutions were investigated by dynamic light scattering (DLS). In the NaCl solutions, we found that strong electrostatic interaction amongBPTI and chloride ions led to the decamer formation only at an acidic pH, whereas the protein solutions were monomeric at a neutral or basic pH, where the electrostatic interaction seemed to be unimportant. The single crystals with a hexagonal truncated bipyramidal shape grew only in the decameric solutions at an acidic pH in the presence of NaCl. On the other hand, the electrostatic interaction was weak in the presence of NaH2PO4/K2HPO4 at all the pH values investigated, and the solutions were always monomeric. In contrast with the NaCl solutions, single crystals with a rod-like shape appeared at a neutral and basic pH in the NaH2PO4/K2HPO4 solutions. These results suggest that the crystallization mechanism in the presence of NaCl, where electrostatic interactions seem to drive crystallization, is totally different from that in the presence of NaH2PO4/K2HPO4 where another interaction such as hydrophobic interaction or hydrogen bonding might cause crystallization. r 2002 Elsevier Science B.V. All rights reserved.