Molecular chaperones assist the folding of newly synthesized proteins during and after the translation in cells. In bacteria, the prevailing view is that Trigger Factor and DnaK/DnaJ chaperones interact with nascent polypeptide co-translationally and GroEL/ES chaperone act mainly after the translation termination. To elucidate the mechanism of these chaperones on the folding of nascent polypeptides, we try to observe the dynamic behavior of chaperones on the translating ribosome at the

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... ecule level. By using TIRF microscopy and reconstituted cell-free translation system (PURE system), we watched the interaction between fluorescence-labeled Trigger Factor and a immobilized translating ribosome. 3P074 一分子蛍光法によるリポアミド脱水素酵素の作用特性の解析 Dihydrolipoamide dehydrogenase (E3) subtracts hydrogen from dihydrolipoamide to reduce NAD to NADH. FAD as an active center of E3 mediates this enzymatic reaction by the periodic redox. Since FAD is fluorescent and the reduced form is non-fluorescent, the fluorescence from single molecule of E3 repeats the periodic transition between the bright and dark states. The duration time and fluorescence intensity of the bright and dark state responding to the enzymatic turn over were analyzed by the home built single molecular fluorescence detection system using a CW laser, fluorescence microscope and MCS. The fluctuation and the cross correlation of two FADs of the enzymatic activity of E3 are reported in detail. Ferritin is a ubiquitous cage-like protein and has a capability to store iron. It is co-polymers of two different subunits (H and L). H subunit has ferroxidase activity, instead of this, L subunit consists of nucleation site of oxidized product. Recombinant L ferritin, fer0, is well studied because it is extremely stable and has potential to synthesize nanoparticles (NPs) of various metal compounds. Although recombinant H ferritin has higher ability for oxidation, it has not been utilized for NPs synthesis, because it is easy to coagulate. To improve NPs synthesis efficiency, we have designed the L-mutant (Y24E) that is one of the essential residues for ferroxidase activity in H subunit. We will report the ability of NPs synthesis in fer0 and the mutant. 3P076 デザインペプチドによる脂肪滴とアミロイド線維の加水分解 Hydrolysis of lipid droplets and amyloid fibrils by the designed peptide Yoshihiro Iida, Atsuo Tamura (Kobe University) Obesity has become a huge problem in modern life. To avoid obesity, we should hydrolyze triglyceride in lipocyte. As a candidate for an anti-obesity drug, we tried to design peptides having enzyme activity like the lipase. As a strategy for the design, we made the peptide to have the catalytic triad composed of histidine, asparagine acid and serine. Based on the strategy, we synthesized 5 peptides named me1-5. It was shown that me5 takes the alpha-helical conformation and can hydrolyze triglyceride. We also tried to hydrolyze amyloid fibrils by using me5, and it was confirmed. We conclude that the peptide me5 can be regarded as a hydrolase which is capable of hydrolyzing the lipid droplets and amyloid fibrils. 3P077 タンパク質分解酵素の速度論的安定性の熱測定による評価 方法 Calorimetric method to evaluate the kinetic stability of proteases Shun-ichi Kidokoro, Akihiro Nagata, Keita Ochi (Dept. Bioengineer., Nagaoka Univ. Tech.) The thermal denaturation of protease is known to be irreversible because of self-digestion. As differential scanning calorimetry (DSC) was a dynamic method in nature, several papers have reported that DSC was useful to evaluate the kinetic stability of proteases. While only the first-order reaction was used in the analysis traditionally, the second-order reaction, self-digestion, becomes apparent in many cases and is introduced in the new model of this study. The concentration dependence of the irreversible thermal denaturation observed by DSC was well explained by the new model, and the kinetic parameters from the model were found to agree well with those determined by other method, analyzing the time course of the remaining enzymatic activity. 3P078 タウタンパク質に対する Pin1 のプロリン異性化活性を測定 するための新しい方法 A novel method to measure Pin1's peptidyl-prolyl isomerase activity for tau protein Teikichi Ikura, Nobutoshi Ito (Med. Res. Inst., Tokyo Med. Dent. Univ.) The Alzheimer's disease-related protein, tau, aggregates into neurofibrillary tangles when it is hyperphosphorylated. A peptidyl-prolyl isomerase (PPIase), Pin1, restored the function of tau by presumably catalyzing isomerization of a specific pSer/Thr-Pro motif. The function of Pin1 for tau, however, is still unsolved due to the lack of methods to measure Pin1's PPIase activity for tau. Here we developed a novel method for this purpose. In this method, catalytic reaction by Pin1 is coupled with dephosphorylation by a trans-isomer-specific phosphatase PP2A. Then the PPIase activity is detected as degree of dephosphorylation by ELISA. In the present study, we demonstrated availability of this method for a synthetic peptide including the pThr231-Pro232 motif of tau.