Creation and structure determination of an artificial protein with three complete sequence repeats

Adachi, Motoyasu; Shimizu, Rumi; Kuroki, Ryota; Blaber, M.

Journal of Synchrotron Radiation, 20(6), p.953 - 957, 2013/11

https://doi.org/10.1107/S0909049513022164

Symfoil-4P is a protein exhibiting the threefold symmetrical beta-trefoil fold designed based on the human acidic fibroblast growth factor. First three asparagine-glycine sequences of Symfoil-4P are replaced with glutamine-glycine (Symfoil-QG) or serine-glycine (Symfoil-SG) sequences protecting from deamidation, and His-Symfoil-II was prepared by introducing a protease digestion site into Symfoil-QG so that Symfoil-II has three complete repeats after removal of the N-terminal histidine tag. The Symfoil-QG and SG and His-Symfoil-II proteins were expressed in as soluble protein, and purified by nickel affinity chromatography. Symfoil-II was further purified by anion-exchange chromatography after removing the HisTag by proteolysis. Symfoil-QG and II crystals gave 1.5 and 1.1, resolution, respectively. The refined crystal structure of Symfoil-II showed pseudo-threefold symmetry as expected from other Symfoils.

Crystal structure of UDP-glucose:anthocyanidin 3--glucosyltransferase from

Hiromoto, Takeshi; Honjo, Eijiro*; Tamada, Taro; Noda, Hisanobu*; Kazuma, Kohei*; Suzuki, Masahiko*; Kuroki, Ryota

Journal of Synchrotron Radiation, 20(6), p.894 - 898, 2013/11

https://doi.org/10.1107/S0909049513020712

Flowers of the butterfly pea () accumulate a group of polyacylated anthocyanins, named ternatins, in their petals. The first step in ternatin biosynthesis is the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin, a reaction catalyzed in by UDP-glucose:anthocyanidin 3--glucosyltransferase (3GT-A; AB185904). To elucidate the structure-function relationship of 3GT-A, recombinant 3GT-A was expressed in and its tertiary structure was determined to 1.85 , resolution by using X-ray crystallography. The structure of 3GT-A shows a common folding topology, the GT-B fold, comprised of two Rossmann-like // domains and a cleft located between the N- and C-domains containing two cavities that are used as binding sites for the donor (UDP-Glc) and acceptor substrates. By comparing the structure of 3GT-A with that of the flavonoid glycosyltransferase GT1 from red grape () in complex with UDP-2-deoxy-2-fluoro glucose and kaempferol, locations of the catalytic His-Asp dyad and the residues involved in recognizing UDP-2-deoxy-2-fluoro glucose were essentially identical in 3GT-A, but certain residues of GT1 involved in binding kaempferol were found to be substituted in 3GT-A. These findings are important for understanding the differentiation of acceptor-substrate recognition in these two enzymes.

Hydrogen-bond network and pH sensitivity in human transthyretin

Yokoyama, Takeshi*; Mizuguchi, Mineyuki*; Nabeshima, Yuko*; Kusaka, Katsuhiro*; Yamada, Taro*; Hosoya, Takaaki*; Ohara, Takashi; Kurihara, Kazuo; Tanaka, Ichiro*; Niimura, Nobuo*

Journal of Synchrotron Radiation, 20(6), p.834 - 837, 2013/11

https://doi.org/10.1107/S090904951302075X

Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade. Low pH is known to promote dissociation into monomer and the formation of amyloid fibrils. In order to reveal the molecular mechanisms underlying pH sensitivity and structural stabilities of TTR, neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. Crystals for the neutron diffraction experiments were grown up to 2.5 mm for four months. The neutron crystal structure solved at 2.0 revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is involved in monomer-monomer and dimer-dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. Structural comparison with the X-ray crystal structure at acidic pH identified the three amino acid residues responsible for the pH sensitivity of TTR. Our neutron model provides insights into the molecular stability related to amyloidosis.

Crystal structure of endo-1,4--glucanase from Eisenia foetida

Arimori, Takao*; Ito, Akihiro*; Nakazawa, Masami*; Ueda, Mitsuhiro*; Tamada, Taro

no journal, , 

Dynamic fluid-structure interaction simulation of high-brightness X-ray generator

Ishiyama, Shintaro

no journal, , 

A prototype thermionic electron gun for a high-brightness X-ray generator has been developed. The X-ray generator is equipped with a newly designed rotating anticathode and aimed towards a maximum brilliance of 60 kWmm. During X-ray generation, the surface of the rotating anticathode has melt down spots due to the electron beam exposure, and these melting spots seems to induce damage of rotor and rotating balance. From view point of safety design toward structural soundness of this system, fluid and structural analysis to the rotor structure is absolutely imperative. In present study, dynamic fluid-structure interaction simulation techniques was applied to high-brightness X-ray generator and stress and deformation behavior during X-ray generation was analyzed.