Katsunori Teranishi

The crystal structure of the photoprotein aequorin at 2.3 A resolution.

Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea. Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium. The aequorin molecule contains four helix–loop–helix ‘EF-hand’ domains, of which three can bind calcium. The molecule also contains coelenterazine as its chromophoric ligand. When calcium is added, the protein complex decomposes into apoaequorin, coelenteramide and CO2, accompanied by the emission of light. Apoaequorin can be regenerated into active aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent. Cloning and expression of the complementary DNA for aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described; however, techniques have only recently been developed to prepare recombinant aequorin of the highest purity, permitting a full crystallographic study. Here we report the structure of recombinant aequorin determined by X-ray crystallography. Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide. The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur.

Light‐emitters involved in the luminescence of coelenterazine

Coelenterazine emits light by chemi-and bioluminescence reactions, decomposing into coelenteramide and CO(2). To ascertain the light emitters involved, the fluorescence of coelenteramide and five analogues were studies in four kinds of solvent. The results showed that coelenteramides can form five kinds of light emitters, ie unionized (lambda(max) 386-423 nm), phenolate anion (lambda(max) 480-490 nm), phenolate anion temporarily formed from the ion-pair state (lambda(max) 465-479 nm), amide anion (lambda(max) 435-458 nm) and pyrazine-N(4) anion (lambda(max) 530-565 nm). The chemiluminescence light emitter of coelenterazine in the presence of alkali (lambda(max) 530-550 nm) was found to be the pyrazine-N(4) anion and not the dianion (ie phenolate anion/amide anion), as previously believed. In chemiluminescence, the normal light emitter is the amide anion, and the pyrazine-N(4) anion emission may occur in the presence of alkali, but light emission from any other emitters has not been observed. In the bioluminescence reaction, the normal light emitter is the amide anion, but no other light emitter was observed except the unionized form found in the Ca-triggered luminescence of semisynthetic aequorins prepared with an e-type coelenterazine instead of coelenterazine.

Adhesion of β-d-glucans to cellulose

Schizophyllan, a Schizophylium communeβ-d-glucan, a Tamarindus xyloglucan, locust bean gum, a galactomannan, a barley β-d-glucan, and chitosan show specific adhesion to microcrystalline cellulose (cellulose I). Xyloglucan, locust bean gum, barley β-d-glucan, and chitosan also show the ability to adhere mercerized cellulose (cellulose II), while schizophyllan does not. As the molecular weight of schizophyllan decreases, both its ability to form triple-helical structures and its adhesion to cellulose I diminish and finally disappear, indicating that the adhesion of schizophyllan to cellulose I depends on high-molecular-weight domains that adopt the triple-helical structures. On the other hand, the adhesion of locust bean gum, chitosan, and xyloglucan to celluloses was found to be largely independent of molecular weight. Furthermore, it is thought that the adhesion of barley β-d-glucan occurs because it belongs to a group of xyloglucans.

The crystal structures of semi-synthetic aequorins

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. Semi‐synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca2+. To understand the structural basis of the Ca2+‐sensitivity, we determined the crystal structures of four semi‐synthetic aequorins (cp‐, i‐, br‐ and n‐aequorins) at resolutions of 1.6–1.8 Å. In general, the protein structures of these semi‐synthetic aequorins are almost identical to native aequorin. Of the four EF‐hand domains in the molecule, EF‐hand II does not bind Ca2+, and the loop of EF‐hand IV is clearly deformed. It is most likely that the binding of Ca2+ with EF‐hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i‐, br‐, and n‐aequorins have bulky 2‐substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca2+ to aequorin. In cp‐aequorin, the cyclopentylmethyl group that substitutes for the original 8‐benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light‐emitting reaction. The differences of various semi‐synthetic aequorins in Ca2+‐sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca2+‐binding.

Efficient Regioselective Synthesis of Mono-2-O-Sulfonyl-cyclodextrins by the Combination of Sulfonyl Imidazole and Molecular Sieves

Abstract Cyclodextrins are cyclic oligosaccharides consisting of six or more α-1,4-linked D-glucopyranose units, which possess primary hydroxyl groups at the C-6 positions and secondary hydroxyl groups at the C-2 and C-3 positions. Because cyclodextrins have a hydrophobic and optically active interior, they have been utilized as transporters of hydrophobic molecules and small molecular mimics of enzymes. The chemical modification of cyclodextrins has been investigated in order to improve these characteristics. Sulfonations of the primary or secondary hydroxyl groups of cyclodextrin have been applied for further functionalization of cyclodextrin, and several methods for regioselective sulfonations have been developed. Among these strategies, selective monotosylation of the C-6 hydroxyl group is done relatively easily by reaction of α or β-cyclodextrin and p-toluenesulfonyl chloride in pyridine1,2 or in alkaline aqueous solution.3,4 However, sulfonation of the secondary hydroxyl groups is more difficult and...

Introduction of Fatty Acids to Starch Granules by Ultra-High-Pressure Treatment

Introduction of fatty acid into maize starch granule by ultra high pressure treatment (UHPT) was investigated. Starches are normal, waxy and amylo-species. They were treated by ball-mill before UHPT. Fatty acids are linoleic, oleic and oleic-stearic acid mixtures. Removal of oil on granule surface was done with 50% ethanol. The incorporated amount of fatty acids and tendency of effect of ball-mill time on the fatty acid amount are different among the three starch species. Fatty acid introduction to amylose and amylopectin helices was also investigated after washing with chloroform-methanol-water mixture. The introduced fatty acid amount to amylostarch is the largest among three species. It is proved that the less double bond in fatty acid, the more fatty acid introduced to starch. The peak at 13.6° (2T) on X-ray diffraction pattern of starch samples treated by UHP becomes large and their Tp on DSC becomes low as increase of introduced fatty acid.

SYNTHESIS OF HYDROPEROXIDE VIA PHOTOOXYGENATION FOR A MODEL AEQUORIN BIOLUMINESCENCE

Abstract Unstable hydroperoxide of coelenterazine (Oplophorus luciferin) analog has been synthesized by the reaction of coelenterazine analog with polymer-bound Rose Bengal via photooxygenation. This compound may be a key intermediate model in the bioluminescence of aequorin and the chemiluminescence of coelenterazine.

Synthesis and enhanced chemiluminescence of new cyclomaltooligosaccharide (cyclodextrin)-bound 6-phenylimidazo[1,2-a]pyrazin-3(7H)-one

In order to provide chemiluminescent substrates that have high light-emitting efficiency in aqueous solution, the structural design on 6-phenylimidazo[1,2-a]pyrazin-3(7H)-one compounds was studied in the covalent attachment of a light-producing chromophore to a cyclomaltooligosaccharide (cyclodextrin). The synthesis of cyclodextrin-bound 6-phenylimidazo[1,2-a]pyrazin-3(7H)-one compounds was achieved by the formation of an amido bond between a 6-phenylimidazo[1,2-a]pyrazin-3(7H)-one and a mono-6-amino-6-deoxycyclodextrin. The properties of oxygen-induced chemiluminescence of the synthesized cyclodextrin-bound light-emitting chromophores were investigated. The light-emitting efficiency in pH 8.3 phosphate buffer was remarkably dependent on the kind of bound cyclodextrin and the binding site between the chromophore and cyclodextrin. The light-emitting efficiency of a cyclodextrin-bound compound in which cyclomaltoheptaose (β-cyclodextrin) had been covalently attached to the 2-position of the imidazo[1,2-a]pyrazin-3(7H)-one ring system showed an up to 11-fold enhancement over that of a non-cyclodextrin chromophore, whereas attachment to cyclomaltohexaose (α-cyclodextrin) resulted in no enhancement. Moreover, this study indicated that the strategy that involves covalently attaching a light-producing chromophore onto a cyclodextrin for the enhancement of chemiluminescence is more efficient than the use of an aqueous solution containing very large amounts of cyclodextrin.

Convenient regioselective mono-2-O-sulfonation of cyclomaltooctaose

Regioselective mono-2-O-sulfonation of cyclomaltooctaose was conveniently achieved by using the combination of sulfonyl imidazole and molecular sieves in DMF. In this reaction, no 3-O- or 6-O-sulfonation products were produced. The reactions do not require strict anhydrous or basic conditions, or specific sulfonyl groups.

Practical and convenient modifications of the A,C-secondary hydroxyl face of cyclodextrins

Abstract A practical and convenient method for the preparation of α-, β-, and γ-cyclodextrin derivatives, in which the secondary hydroxyl faces of A- and C-glucose units are regioselectively modified, has been developed. Reactions of α-, β-, and γ-cyclodextrins with 1,4-dibenzoylbenzene-3′,3″-disulfonyl imidazole in N , N -dimethylformamide in the presence of molecular sieves regioselectively afforded the corresponding cyclic 2 A ,2 C -(1,4-dibenzoylbenzene-3′,3″-disulfonyl)-cyclodextrins. Subsequent treatment of the sulfonylated cyclodextrins with sodium hydroxide or aqueous ammonia afforded the corresponding 2 A ,3 A :2 C ,3 C -di-manno-epoxy-cyclodextrins or 3 A ,3 C -diamino-3 A ,3 C -dideoxy-(2 A S,2 C S,3 A S,3 C S)-cyclodextrins, respectively, which can serve as important intermediates for further functionalizations of the cyclodextrins.