Biomedical subjects

Takashi Morii

Publications and source records attributed to Takashi Morii.

24 records · Page 2Linked to original sources

PubMed2002

In vitro selection of ATP-binding receptors using a ribonucleopeptide complex.

A recently described three-dimensional structure of the ribosome provides a sense of remarkable diversity of RNA-protein complexes. We have designed a new class of scaffold for artificial receptors, in which a short peptide and RNA with a randomized nucleotide region form a stable and specific complex. 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 directly that the peptide is incorporated in the functional structure of RNA and suggest that amino acids outside the RNA-binding region of the peptide modulate the ATP-binding of ribonucleopeptide. Our approach would provide an alternative strategy for the design of "tailor-made" ribonucleopeptide receptors and enzymes.

Adenosine Triphosphate↗

PubMed2002

A new fluorescent biosensor for inositol trisphosphate.

An intracellular second messenger d-myo-inositol-1,4,5-trisphosphate (IP3) is a key biological signaling molecule that controls the cellular Ca2+ concentration. We report the preparation and evaluation of a functionalized protein-based sensor for IP3 by exploring the selective IP3 binding properties of pleckstrin homology (PH) domain. Signal transduction is imparted to the protein by mutation of proximal residues to cysteine and then alkylation of the active site by various fluorophore derivatives. This creates functionalized proteins that show micromolar affinity for IP3, reasonably strong fluorescence emission, and wavelength changes in the fluorophore and selectivity higher than the original PH domain among different inositol phosphate derivatives.

Amino Acid Sequence↗

PubMed2002

Structure-based design of a leucine zipper protein with new DNA contacting region.

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.

Amino Acid Sequence↗

PubMed2002

A general strategy to determine a target DNA sequence of a short peptide: application to a d-peptide.

Short peptides could potentially provide a novel element to read-out DNA sequences from the major groove. However, it is difficult to determine sequence-preference of de novo designed monomeric short peptides. Because DNS-binding affinity and specificity of short peptides are usually much lower than those of native DNA-binding proteins, determining the sequence-preference of short peptides by conventional methods utilized to deduce the target sequence of proteins often produces an unclear outcome. We report here a general strategy to defining the sequence-preference of a DNA-binding short peptide by using the heterodimers. A GCN4 basic region peptide tethers a low-affinity DNA-binding peptide adjacent to a GCN4 binding sequence through the cyclodextrin-adamantane association, thereby increasing local concentration of the low-affinity peptide on degenerated DNA sequences. An increase of the local concentration allows one to select a preferential sequence for the low-affinity DNA binding peptide. The method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of d-amino acids. The method is potentially applicable not only to artificial peptides, but also to other synthethic ligands.

Amino Acid Sequence↗

PubMed2002

Formation of a fairly stable diazoate intermediate of 5-methyl-2'-deoxycytidine by HNO2 and NO, and its implication to a novel mutation mechanism in CpG site.

The intermediate produced from 5-methyl-2'-deoxycytidine ((5me)dCyd) by HNO2 and NO treatments was isolated and characterized. When 10mM (5me)dCyd was incubated with 100mM NaNO2 at pH 3.7 and 37 degrees C, a previously unidentified product was formed. The product was identified as a diazoate derivative of (5me)dCyd, 1-(beta-D-2'-deoxyribofuranosyl)-5-methyl-2-oxopyrimidine-4-diazoate ((5me)dCyd-diazoate), on the bases of several measurements including LC/MS. The time course of the concentration change of the diazoate showed a characteristic profile of a reaction intermediate, and the steady state concentration was 2.3 microM (0.023% yield). When an aqueous solution of 10mM (5me)dCyd (10 mL) was bubbled by NO at 37 degrees C under aerobic conditions holding the pH around 7.4, the diazoate was also generated. The yield of the diazoate was 0.041 micromol (0.041% yield) at 20 mmol of NO absorption. At physiological pH and temperature (pH 7.4, 37 degrees C), the diazoate was converted to dThd exclusively with a first order rate constant k=9.1x10(-6) x s(-1) (t(1/2)=21 h). These results show that the diazoate is generated as a relatively stable intermediate in the reactions of (5me)dCyd with HNO2 and NO and further suggest that the diazoate can be formed in cellular DNA with biologically relevant doses of HNO2 and NO.

Azo Compounds↗

PubMed2002

A new strategy to determine the target DNA sequence of a short peptide.

The design of molecules that target desired DNA sequences has been one of the major challenges in the field of molecular recognition. We report here a general strategy for defining the sequence-preference of DNA-binding short peptide by using its heterodimer. Our method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of D-amino acids.

Base Sequence↗