Online ISSN : 1349-2942
ISSN-L : 1349-2942
Volume 6
Displaying 1-7 of 7 articles from this issue
Regular Article
  • Takaaki Sato, Togo Shimozawa, Toshiko Fukasawa, Masako Ohtaki, Kenji A ...
    2010 Volume 6 Pages 1-11
    Published: 2010
    Released on J-STAGE: January 21, 2010
    Supplementary material
    Using small-angle X-ray scattering (SAXS), we have studied the initial stage (nucleation and oligomerization) of actin polymerization induced by raising temperature in a stepwise manner from 1°C to 30°C at low ionic strength (4.0 mg ml–1 actin in G-buffer). The SAXS experiments were started from the mono-disperse G-actin state, which was confirmed by comparing the scattering pattern in q- and real space with X-ray crystallographic data. We observed that the forward scattering intensity I(q→0), used as an indicator for the extent of polymerization, began to increase at ~14°C for Mg-actin and ~20°C for Ca-actin, and this critical temperature did not depend on the nucleotide species, i.e., ATP or ADP. At the temperatures higher than ~20°C for Mg-actin and ~25°C for Ca-actin, the coherent reflection peak, which is attributed to the helical structure of F-actin, appeared. The pair-distance distribution functions, p(r), corresponding to the frequency of vector lengths (r) within the molecule, were obtained by the indirect Fourier transformation (IFT) of the scattering curves, I(q). Next, the size distributions of oligomers at each temperature were analyzed by fitting the experimentally obtained p(r) with the theoretical p(r) for the helical and linear oligomers (2–13mers) calculated based on the X-ray crystallographic data. We found that p(r) at the initial stage of polymerization was well accounted for by the superposition of monomer, linear/helical dimers, and helical trimer, being independent of the type of divalent cations and nucleotides. These results suggest that the polymerization of actin in G-buffer induced by an increase in temperature proceeds via the elongation of the helical trimer, which supports, in a structurally resolved manner, a widely believed hypothesis that the polymerization nucleus is a helical trimer.
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  • Tatsuhito Matsuo, Yutaka Ueno, Yasunori Takezawa, Yasunobu Sugimoto, T ...
    2010 Volume 6 Pages 13-26
    Published: 2010
    Released on J-STAGE: February 23, 2010
    In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ~6 Å and rotated by ~16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ~12° and radially by ~7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.
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  • Koro Nishikata, Sotaro Fuchigami, Mitsunori Ikeguchi, Akinori Kidera
    2010 Volume 6 Pages 27-36
    Published: 2010
    Released on J-STAGE: March 16, 2010
    Supplementary material
    The halobacterial transducer of sensory rhodopsin II (HtrII) is a photosignal transducer associated with phototaxis in extreme halophiles. The HAMP domain, a linker domain in HtrII, is considered to play an important role in transferring the signal from the membrane to the cytoplasmic region, although its structure in the complex remains undetermined. To establish the structural basis for understanding the mechanism of signal transduction, we present an atomic model of the structure of the N-terminal HAMP domain from Natronomonas pharaonis (HtrII: 84–136), based on molecular dynamics (MD) simulations. The model was built by homology modeling using the NMR structure of Af1503 from Archaeoglobus fulgidus as a template. The HAMP domains of Af1503 and HtrII were stable during MD simulations over 100 ns. Quantitative analyses of inter-helical packing indicated that the Af1503 HAMP domain stably maintained unusual knobs-to-knobs packing, as observed in the NMR structure, while the bulky side-chains of HtrII shifted the packing state to canonical knobs-into-holes. The role of the connector loop in maintaining structural stability was also discussed using MD simulations of loop deletion mutants.
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  • Tomoki Kitagawa, Noriaki Murakami, Seido Nagano
    2010 Volume 6 Pages 37-51
    Published: 2010
    Released on J-STAGE: March 19, 2010
    Pancreatic β-cells are interconnected by gap junctions, which allow small molecules to pass from cell to cell. In spite of the importance of the gap junctions in cellular communication, modeling studies have been limited by the complexity of the system. Here, we propose a mathematical gap junction model that properly takes into account biological functions, and apply this model to the study of the β-cell cluster. We consider both electrical and metabolic features of the system. Then, we find that when a fraction of the ATP-sensitive K+ channels are damaged, robust insulin secretion can only be achieved by gap junctions. Our finding is consistent with recent experiments conducted by Rocheleau et al. Our study also suggests that the free passage of potassium ions through gap junctions plays an important role in achieving metabolic synchronization between β-cells.
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  • Masanobu Sakata, Tamami Kawasaki, Toshimichi Shibue, Hideo Namiki
    2010 Volume 6 Pages 53-57
    Published: 2010
    Released on J-STAGE: December 07, 2010
    Ferromagnetic resonance (FMR) with an electron spin resonance (ESR) apparatus was investigated for superparamagnetic particles within Daphnia resting eggs. High-field (HF) resonance lines near g=2 resulted from single superparamagnetic particles, were detected from ESR spectra of Daphnia resting eggs. The size of isolated superparamagnetic particles within Daphnia resting eggs was calculated to be approximately 13 nm in diameter by analysis of the temperature dependence of the HF line width. Small-angle X-ray scattering (SAXS) analysis of Daphnia resting eggs also showed that average size of superparamagnetic particles in diameter, equivalent to magnetite, was approximately 13 nm. The combination of FMR and SAXS measurement is very effective in estimating the size of superparamagnetic particles in biological organisms, with difficulties of preparing for samples for measurement by electron microscopy. However, Chlorella, with that Daphnia were raised, did not show FMR spectra, showing no magnetic particles within Daphnia resting eggs. Therefore, it suggested that superparamagnetic particles within Daphnia resting eggs, were mineralized in Daphnia as the result of biomineralization of Fe originated from Chlorella.
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  • Takamitsu Haruyama, Yoko Hirono-Hara, Yasuyuki Kato-Yamada
    2010 Volume 6 Pages 59-65
    Published: 2010
    Released on J-STAGE: December 17, 2010
    The F1-ATPase, the soluble part of FoF1-ATP synthase, is a rotary molecular motor consisting of α3β3γδε. The γ and ε subunits rotate relative to the α3β3δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F1-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F1-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α3β3γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.
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  • Hiroshi C. Watanabe, Yoshiharu Mori, Takashi Tada, Shozo Yokoyama, Tak ...
    2010 Volume 6 Pages 67-78
    Published: 2010
    Released on J-STAGE: December 25, 2010
    Supplementary material
    The synergetic effects of multiple rhodopsin mutations on color tuning need to be completely elucidated. Systematic genetic studies and spectroscopy have demonstrated an interesting example of synergetic color tuning between two amino acid residues in conger rhodopsin's ancestral pigment (p501): — a double mutation at one nearby and one distant residue led to a significant λmax blue shift of 13 nm, whereas neither of the single mutations at these two sites led to meaningful shifts.
    To analyze the molecular mechanisms of this synergetic color tuning, we performed homology modeling, molecular simulations, and electronic state calculations. For the double mutant, N195A/A292S, in silico mutation analysis demonstrated conspicuous structural changes in the retinal chromophore, whereas that of the single mutant, A292S, was almost unchanged. Using statistical ensembles of QM/MM optimized structures, the excitation energy of retinal chromophore was evaluated for the three visual pigments. As a result, the λmax shift of double mutant (DM) from p501 was −8 nm, while that of single mutant (SM) from p501 was +1 nm. Molecular dynamics simulation for DM demonstrated frequent isomerization between 6-s-cis and 6-s-trans conformers. Unexpectedly, however, the two conformers exhibited almost identical excitation energy, whereas principal component analysis (PCA) identified the retinal-counterion cooperative change of BLA (bond length alternation) and retinal-counterion interaction lead to the shift.
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