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Biomedical subjects

Itaru Hamachi

Publications and source records attributed to Itaru Hamachi.

28 records · Page 2Linked to original sources
PubMed2004

Recognition and fluorescence sensing of specific amino acid residue on protein surface using designed molecules.

Many biological processes are mediated by surface recognition between proteins. Small molecules that recognize and bind a specific region of a protein surface may be promising agents for disrupting certain protein-protein surface interactions, which consequently leads to regulation of cellar functions. This article describes our recent efforts toward the development of the designed small molecules, which can recognize histidine or phosphorylated amino acid residues on peptide surfaces in a sequence-selective manner. These results demonstrate that cooperative metal-ligand interaction is powerful for tight and selective binding to the specific amino acid residues of proteins in aqueous medium.

Amino Acid Sequence↗
PubMed2003

Semi-wet peptide/protein array using supramolecular hydrogel.

The protein microarray is a crucial biomaterial for the rapid and high-throughput assay of many biological events where proteins are involved. In contrast to the DNA microarray, it has not been sufficiently established because of protein instability under the conventional dry conditions. Here we report a novel semi-wet peptide/protein microarray using a supramolecular hydrogel composed of glycosylated amino acetate. The spontaneous gel-formation and amphiphilic properties of this supramolecular hydrogel have been applied to a new type of peptide/protein gel array that is compatible with enzyme assays. Aqueous cavities created in the gel matrix are a suitable semi-wet reaction medium for enzymes, whereas the hydrophobic domains of the fibre are useful as a unique site for monitoring the reaction. This array system overcomes several drawbacks of conventional protein chips, and thus can have potential applications in pharmaceutical research and diagnosis.

Equipment Design↗
PubMed2003

Cross-linking strategy for molecular recognition and fluorescent sensing of a multi-phosphorylated peptide in aqueous solution.

In the research field of molecular recognition, selective recognition and sensing of phosphorylated protein surfaces is strongly desirable both for elucidation of protein-protein recognition at the molecular level and for regulation of signal transduction through protein surfaces. Here we describe a new strategy for molecular recognition of a multi-phosphorylated peptide using intrapeptide cross-linking on the basis of coordination chemistry. The present artificial receptor can selectively bind to doubly phosphorylated peptide through multiple-point interactions and fluorescently sense the binding event with an association constant of more than 106 M-1 in neutral aqueous solution.

Circular Dichroism↗
PubMed2003

Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors.

A new general method, post-photoaffinity-labeling modification (PPALM), for constructing fluorescent saccharide biosensors based on naturally occurring saccharide-binding proteins, lectins, is described in detail. An active-site-directed incorporation of a masked reactive site into a lectin was conducted by using a photoaffinity labeling technique followed by demasking and then chemical modification to yield a fluorescent lectin. Two photoaffinity labeling reagents were designed and synthesized in this study. The labeling reagent with a photoreactive site appended through a disulfide link to a mannoside unit was bound to the saccharide-binding pocket of the lectin concanavalin A (Con A). After light irradiation, the mannoside unit was cleaved by reduction. The unique thiol group thus produced was site-specifically modified with various fluorescent groups (dansyl, coumarin, or dimethylaminobenzoate derivatives) to afford fluorescent Con As. The labeling site was characterized by protease-catalyzed digestion followed by HPLC, MALDI-TOF MS, and tandem mass-mass spectrometry; these methods indicated that the photolabeling step is remarkably site specific. Strong fluorescence was observed in the engineered Con A with a fluorophore, and the emission changed sensitively upon saccharide complexation. The binding constants for various saccharides were determined by fluorescence titration and demonstrated that the binding selectivity and affinity of the engineered Con As are comparable to those of native Con A. The red shift of the emission maximum, the decrease in the fluorescence anisotropy of the dansyl unit, and the increase in the twisted intramolecular charge transfer emission caused by sugar binding to the engineered Con A explicitly indicate that the microenvironment of the appended fluorophores changes from a restricted and relatively hydrophobic environment into a rather freely mobile and hydrophilic environment.

Binding Sites↗
PubMed2003

Combinatorial library of low molecular-weight organo- and hydrogelators based on glycosylated amino acid derivatives by solid-phase synthesis.

A combinatorial approach for the synthesis of supramolecular gelators as new organic materials is described herein. In the course of the development of a convenient and flexible solid-phase synthesis of the artificial glycolipids, some of these compounds were accidentally found to act as low molecular-weight gelators toward organic solvents. Using this combinatorial solid-phase synthesis of glycosylated amino acetates, screening and optimization of low molecular-weight organo/hydro-gelators were efficiently carried out. We found that an N-acetyl-galactosamine-appended amino acid ester (GalNAc-aa) efficiently gelates a broad spectrum of organic solvents. More interestingly, some GalNAc-aa derivatives displayed an excellent hydrogelation capability. Transmission electron microscopy, scanning electron microscopy, confocal laser scanning microscopy, and FT-IR were used for characterization of the gel structure. It is indicated that supramolecular fibers supported by strong hydrogen-bonding networks are entangled so that the resulting spaces can immobilize a number of solvent molecules effectively. In addition, the supramolecular hydrogel consisting of GalNAc-suc-glu(O-methyl-cyc-pentyl)(2) is stable even under high salt concentrations probably due to its nonionic character and as a result, a native protein is successfully entrapped in the gel matrix without denaturation.

Amino Acids↗
PubMed2002

Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9).

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man(8) and Man(9). Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manalpha(1-2)Manalpha (which represents the terminal disaccharide of all three arms of Man(9)) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manalpha(1-2)Manalpha complex revealed that CVN recognizes the stacked conformation of Manalpha(1-2)Manalpha through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manalpha(1-2)Manalpha(1-2)Man, methyl Manalpha(1-2)Manalpha(1-3)Man and methyl Manalpha(1-2)Manalpha(1-6)Man with remarkable selectivity, and binds these trisaccharides with K(A) values ranging from 8.1x10(3)M(-1) to 6.6x10(6)M(-1). Site 2 is less selective in that it binds all three trisaccharides with similar K(A) values ranging from 1.7 to 3.7(+/-0.3)x10(5)M(-1), but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display alpha(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source.

Amino Acid Sequence↗
PubMed2002

First thermally responsive supramolecular polymer based on glycosylated amino acid.

In materials science, a dynamic property sensitive to an environmental change (heat, light, electric current, pH, and other chemical or physical changes) is indispensable for intelligent materials. Such organic materials, however, are very limited even in conventional polymers. This paper clearly demonstrates that, regardless of the low molecular weight, a glycosylated amino acid derivative newly screened by a combinatorial method forms a macroscopic supramolecular hydrogel that reversibly swells or shrinks in response to the external temperature. Using the unique thermal response of the present hydrogel, we carried out the controlled release of DNA and the perfect removal of bisphenol A from the polluted water. Recently, advanced supramolecular polymers, in which monomers are noncovalently connected, are expected to be highly advantageous over traditional polymers because of their tunable and recyclable characteristics. The present result newly confers a dynamic feature on the supramolecular polymers, which is desirable for the sophisticated application in many fields.

Acetylgalactosamine↗
PubMed2002

First artificial receptors and chemosensors toward phosphorylated peptide in aqueous solution.

The first fluorescent chemosensors toward a native phosphorylated peptide are successfully synthesized. Dinuclear zinc(II)-dipicolylamine-based anthracene (1, 2) can selectively recognize and sense phosphorylated species with an increase in the fluorescence intensity. We also demonstrated that these artificial receptors fluorometrically detect a phosphorylated peptide with high affinity (>107 M-1) in aqueous solution.

Amines↗
PubMed1998

Design and Semisynthesis of Photoactive Myoglobin Bearing Ruthenium Tris(2,2'-bipyridine) Using Cofactor-Reconstitution.

A new strategy for semisynthesis of a photoactivatable redox protein is described. Three protohemin molecules with ruthenium tris(2,2'-bipyridine) attached by different spacers were synthesized. The Ru(bpy)(3)-protohemins were incorporated into the heme crevice of apomyoglobin (apo-Mb) to yield semisynthetic Mbs carrying Ru(bpy)(3) as a photosensitizer (Ru(bpy)(3)-Mb). The photoactivation properties and the reaction mechanisms of Ru(bpy)(3)-Mbs were investigated by steady-state photoirradiation and laser flash photolysis. The photoactivation of Ru(bpy)(3)-Mbs was spectrophotometrically demonstrated by comparison with an intermolecular control, namely an equimolar mixture of Ru(bpy)(3) and native Mb. The spacer structure considerably influenced net activation efficiency over a wide pH range as measured by steady-state visible light irradiation and quantum yield. Laser flash photolysis yielded the rate of the photoinduced electron transfer (ET) from the lifetime of the excited Ru(bpy)(3) (k(et) = 4.4 x 10(7) s(-)(1) for Mb(1b) and k(et) = 3.7 x 10(7) s(-)(1) for Mb(1c)) and the back ET rate (k(back) = (2.0-3.7) x 10(7) s(-)(1) for Mb(1b) and k(back) = (1.4-2.4) x 10(7) s(-)(1) for Mb(1c)) from the decay of the transient absorption. These data consistently explained the results of the net photoreaction as follows. (i) The intermolecular control system was less photoactivated because little ET occurred from the excited state of Ru(bpy)(3) to Mb. (ii) The short lifetime of the charge-separated state after photoinduced ET greatly decreased the photoactivation efficiency of Ru(bpy)(3)-Mb with the shortest spacer. (iii) The photochemical and photophysical data of the other two Ru(bpy)(3)-Mb derivatives (the net photoreaction, quantum yield, and ET/back ET rates) were essentially identical, indicating that flexible spacers consisting of oxyethylene units do not rigidly fix the distance between Ru(bpy)(3) and the heme center of Mb. In addition, Ru(bpy)(3)-Mbs were highly photoactivated under aerobic conditions in a manner similar to that under anaerobic conditions, although O(2) usually quenches the photoexcited state of Ru(bpy)(3). This was probably due to the accelerated intramolecular ET from Ru(bpy)(3) to heme, not to O(2) in Ru(bpy)(3)-Mbs. We therefore showed that visible light affects the content of O(2)-bound Mb even in air.

Journal Article↗