Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program.CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad-spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram-negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram-negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α-helical structure in a solution containing LPS. For NMR experiments, we expressed 15 N-and 13 C-labeled CP1 in bacterial cells and successfully assigned almost all backbone and side-chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr-NOE) experiments in LPS. We performed 15 N-edited and 13 C-edited Tr-NOE spectroscopy (Tr-NOESY) for CP1 bound to LPS. Tr-NOE peaks were observed at the only Cterminal region of CP1 in LPS. The results of structure calculation indicated that the Cterminal region (Lys15-Gly29) formed the well-defined α-helical structure in LPS.Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at GlcN I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A.

Epidemiological studies have suggested that alternating magnetic fields affect human body, but mechanism of influences on cell or human body has not established in the biological experiment yet. In order to reveal primary site of action by alternating magnetic fields, we focus on ferritin, iron-storage protein, because ferritin is source of iron ions which produce reactive oxygen species. We exposed ferritin solution to alternating magnetic fields (50Hz~500kHz for 3 h) and measured the concentration of iron ions outside ferritin. The results show that concentration of iron ions is decreased after the exposure. We are currently performing the experiment under wider conditions and will report the detailed analysis of this phenomenon. Nitrile Hydratase (NHase) catalyzes hydration of nitriles to the corresponding amides and has been used for industrial productions of various amides. The active site of NHase consists of an Fe(III) or a Co(III) ion coordinated with two carboxamido nitrogens, one cysteine-sulfer (Cys-S), one cysteine-sulfenic (Cys-SO) and one cysteine-sulfinic (Cys-SO2) moieties. Several reaction paths have been proposed for the catalytic mechanism of NHase, and recently quantum chemical calculations using active-site models were reported (Hopmann et al., 2007(Hopmann et al., , 2008(Hopmann et al., , and 2014. 1P074 ニトリルヒドラターゼ（NHase）によるアミド生成機構に関 する理論的研究In the present study, we have examined four reaction mechanisms by using Quantum Mechanics/Molecular Mechanics (QM/MM) method to reveal the effects of surrounding protein environment on the reaction process. The chaperonin GroE encapsulates a substrate in its inner cavity, assisting the folding in an ATP-dependent manner. Although the function of GroEL has been well characterized, the role of its C-terminal region remains elusive. Here, we investigated the effect of C-terminal truncation on the GroEmediated folding of GFP. We found that the yield of an in-cage folding mediated by a single-ring GroEL was significantly decreased by truncation. In contrast, the yield of an in-cage folding mediated by an ATPasedeficient double-ring GroEL, which formed a stable football shaped complex, was not affected by truncation. These results suggest that the Cterminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. Antimicrobial peptides (AMPs) are ubiquitously found in living organisms and has a broad antimicrobial activity against Gram-negative and Grampositive bacteria and fungi. Some AMPs can bind to lipopolysaccharide (LPS) that is a major component of the outer membrane of Gram-negative bacteria and cause fatal septic shock. Therefore, AMPs that interact with LPS are expected to be novel antibiotics for sepsis. Tachyplesin I (TP I) is one of the AMPs that binds to LPS, but complex structure between TP I and LPS and binding mechanism are still unknown. We investigated the NMR structure of TP I in the presence of LPS and determined the complex structure using docking program. It was indicat...

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