Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches

ALG-2 is a penta-EF-hand Ca(2+)-binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, PXYPXnYP (X, variable; n = 4), in ALIX and PLSCR3; type 2, PXPGF, in Sec31A and PLSCR3. The previously solved X-ray crystal structure of the complex between ALG-2 and an ALIX peptide containing type 1 motif showed that the peptide binds to Pocket 1 and Pocket 2. Co-crystallization of ALG-2 and type 2 motif-containing peptides has not been successful. To gain insights into the molecular basis of type 2 motif recognition, we searched for a new hydrophobic cavity by computational algorithms using MetaPocket 2.0 based on 3D structures of ALG-2. The predicted hydrophobic pocket designated Pocket 3 fits with N-acetyl-ProAlaProGlyPhe-amide, a virtual penta-peptide derived from one of the two types of ALG-2-binding sites in PLSCR3 (type 2 motif), using the molecular docking software AutoDock Vina. We investigated effects of amino acid substitutions of the predicted binding sites on binding abilities by pulldown assays using glutathione-S-transferase -fused ALG-2 of wild-type and mutant proteins and lysates of cells expressing green fluorescent protein -fused PLSCR3 of wild-type and mutants. Substitution of either L52 with Ala or F148 with Ser of ALG-2 caused loss of binding abilities to PLSCR3 lacking type 1 motif but retained those to PLSCR3 lacking type 2 motif, strongly supporting the hypothesis that Pocket 3 is the binding site for type 2 motif.     

Two-dimensional single-shot tomography using a virtually imaged phased array and a spatial phase modulator

A virtually imaged phased array (VIPA) installed optical interferometer has been expanded to the two-dimensional (2D) tomography from the previous one-dimensional single-shot imaging technique with keeping the resolution and the measurement range. A single-shot measurement has been realized by a spatial phase modulator installed in the optical interferometer and tracing the delay time to pixel numbers on a 2D charge-coupled device (CCD) image sensor. The flexibility of the sample position was experimentally confirmed to be >25 mm, in relation to the VIPA coherency, for which the number of the interference order was confirmed to be 35. As a demonstration, a surface profile of stacked gauge blocks was observed. The repeatability of the surface position was 5?μm for the surface profilometry. In addition, a multilayer structure was observed using a glass plate. The experimental resolution was 53?μm when the amplified spontaneous emission light generated by the optical fiber amplifier was used for the light source. The single-shot measurement was confirmed by the 2D-CCD at a frame rate of 30 frames per second (FPS), and it provided evidence that the 2D scanless profilometry was successfully achieved using the VIPA optical device.     

Design study of the demonstration FBR in Japan

The objective of the plant design study Phase 2, conducted by the Japan Atomic Power Company since 1997 for 3 years, is to accomplished a plant overall concept of the Demonstrative FBR (DFBR) that has economical potential toward commercialization and offers high reliability to plant operators not to cause a long unexpected shutdown resulting from a trouble, i.e., sodium leakage or fires. This has been successfully achieved by establishment of a plant overall design of 672 MWe consisting of the reactor system with drastically simplified internals, the compact and double walled coolant boundaries, the well rationalized fuel handling system, the BOP systems introducing up-to-date LWR equipment, and the compact reactor building.

The plant construction cost has been estimated based on the quantity of materials to be about 130 % on the bases of a 1000 MW LWR, which is well contented with the requirement.

The DFBR plant concept, having economical potential toward commercialization, safety and reliability, has been established in the plant design study Phase 2.