Biomedical subjects

Katsutoshi Ohkubo

Publications and source records attributed to Katsutoshi Ohkubo.

10 recordsLinked to original sources

PubMed2005

Dual DNA recognition codes of a short peptide derived from the basic leucine zipper protein EmBP1.

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a beta-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5'-GCCACGTGGC-3' and the native GCN4 binding 5'-ATGACGTCAT-3' sequences with almost equal affinity in the alpha-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5'-ATGAC-3' in the alpha-helical conformation, but it neither shows affinity nor helix formation with 5'-GCCAC-3'. Because native EmBP1 forms 100 times more stable complex with 5'-GCCACGTGGC-3' over 5'-ATGACGTCAT-3', our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.

Amino Acid Sequence↗

PubMed2005

Stepwise molding of a highly selective ribonucleopeptide receptor.

The structural characteristics of RNA-peptide (RNP) complexes are suitable for molding of a ligand-binding pocket of the RNP complex in a stepwise manner. The first step involves molding of the RNA subunit by in vitro selection of an RNP pool originating from an RNA library and the peptide, as previously reported for the construction of an ATP-binding RNP complex from an RRE RNA-Rev peptide complex. The second step involves selection from an RNP library consisting of Rev peptides with randomized amino acid residues and the RNA subunit selected in the first molding. The ATP-binding pocket produced by sequential molding of RNA and peptide subunits shows higher affinity to ATP and a distinct specificity for ATP versus dATP as compared to the ATP-binding RNP receptor in which only the RNA subunit has been molded. The second step selection from the peptide-based RNP library allows expansion of the ATP recognition surface, consisting of both RNA and peptide subunits, to enhance the affinity and selectivity to discriminate ATP against dATP. Our approach of stepwise molding offers the advantage of increasing the diversity of the RNP library by utilizing characteristics of different biopolymers. The ribonucleopeptide-based, multi-subunit approach is also extendable to other biomacromolecular assemblies, which may yield artificial receptors and enzymes with increased specificity and more diverse chemical activities.

Adenosine Triphosphate↗

PubMed2004

Photosensitized NADH formation system with multilayer TiO2 film.

A TiO(2)/polymer film on a quartz plate fabricated by a layer-by-layer method was employed for NADH production from NAD(+) with lipoamide dehydrogenase and methyl viologen in Tris-HCl buffer on irradiation with UV light, and the ultra thin film prevented from enzyme deactivating effectively.

Buffers↗

PubMed2004

Ribonucleopeptides: functional RNA-peptide complexes.

Selection of functional RNAs from randomized pool of RNA molecules successfully affords RNA aptamers that specifically bind to small molecules, and that have catalytic activities. Recent structural analyses of the ribosomal RNA complex suggest that the RNA-protein complex would be a new structural candidate for the design of tailor-made receptors and enzymes. We have designed an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and successive in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. The randomized nucleotide region was placed next to the HIV-1 Rev response element to enable the formation of "ribonucleopeptide" pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP-binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers, and completely lost its ATP-binding ability in the absence of the Rev peptide. The ATP-binding activity of the ribonucleopeptide was increased by a substitution of the N-terminal amino acid of the Rev peptide. These results demonstrate that the peptide stabilizes the functional structure of RNA and suggest that amino acids outside the RNA binding region of the peptide participate in the ATP binding. Our approach would provide a new strategy for the design of tailor-made ribonucleopeptide receptors.

Adenosine Triphosphate↗

PubMed2004

Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells.

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.

Animals↗

PubMed2004

Design of sensing ribonucleopeptides for small ligands.

Here we report a simple method to convert synthetic receptors to fluorescent sensors. An RNA-peptide complex (ribonucleopeptide) with a known three-dimensional structure is used as a framework of the receptor. Artificial ribonucleopeptide sensors were created with a combination of in vitro selection method and successive modification of the peptide with a fluorophore. A ribonucleopeptide complex of the fluorophore labeled peptide showed a remarkable fluorescence emission change upon binding cognate ligands.

Amines↗

PubMed2004

The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase.

A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 degrees C of the variant was 1.7-fold higher than that of the wild type. The K(m) values of the mutant at 37 degrees C were 0.40 mM for NAD(+) and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD(+) and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 degrees C, and 47 degrees C, respectively. The thermal stability of the mutant was lower than that of the wild type.

Aldehyde Oxidoreductases↗

PubMed2003

Functional reassembly of a split PH domain.

The pleckstrin homology (PH) domain forms a structurally conserved protein module of approximately 120 amino acid residues. Several proteins involved in cellular signaling and cytoskeletal organization possess split PH domains while their biological roles and ligand binding activity remain to be clarified. We have designed a split PH domain from a structurally well-characterized PH domain of phospholipase Cdelta(1) by dissecting the PH domain and tethering a coiled coil module to each subunit to ask a question of whether the coiled coil could mediate a functional reassembly of the split PH domain. Isothermal titration microcalorimetry measurements indicated a formation of a thermodynamically stable 1:1 complex of the N-terminal and C-terminal halves of the split PH domain by the coiled coil formation. The reassembled split PH domain binds to IP(3), a target molecule of the parent PLCdelta(1) PH domain, but not to L-IP(3), indicating that the split PH domain maintains a binding selectivity similar to the native PLCdelta(1) PH domain. These results demonstrate that the split PH domain folds into a functional structure when the split halves are brought to close proximity, and suggest that the native split PH domains, such as found in PLCgamma(1), have distinctive functions upon the reassembly.

Amino Acid Sequence↗

PubMed2003

Design of a ribonucleopeptide biosensor.

Ribonucleopeptide receptors for ATP have been designed by using a structure-based design and in vitro selection method. The ATP binding ribonucleopeptide receptors revealed submillimolar affinity to ATP and discriminate ATP against other ribonucleotides. In this research, we have developed a simple strategy to convert the ATP-binding ribonucleopeptide receptor into a ribonucleopeptide sensor by introducing a fluorophore in the peptide subunit. Fluorophore labeled ribonucleopeptide complex showed a large change in the fluorescence intensity upon addition of ATP.

Adenosine Triphosphate↗

PubMed2003

Optimization of an ATP-binding ribonucleopeptide receptor.

We describe here a new strategy for the selection and evolution of functional RNA-peptide complexes. An ATP-binding ribonucleopeptide was obtained by means of a structure-based design of ribonucleopeptide domain and by in vitro selection of the RNA subunit of the ribonucleopeptide. Approaches to optimize the ligand-binding selectivity of ribonucleopeptide will be discussed.

Adenosine Triphosphate↗