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Hisakazu Mihara

Publications and source records attributed to Hisakazu Mihara.

29 records · Page 2Linked to original sources
PubMed2004

Peptide arrays with designed alpha-helical structures for characterization of proteins from FRET fingerprint patterns.

A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.

Amino Acid Sequence↗
PubMed2003

Fluorescence resonance energy transfer in a novel cyclodextrin-peptide conjugate for detecting steroid molecules.

A novel cyclodextrin conjugated peptide, 1, having two different fluorophores, coumarin and pyrene, in the side chains has been designed and synthesized. The circular dichroism study reveals that 1 shows typical alpha-helix pattern, and forms intramolecular inclusion complex with coumarin. The fluorescence emission study shows that the peptide exhibits intramolecular fluorescence resonance energy transfer (FRET) without quenching of two fluorophores. We have determined the binding constants of 1 for various biologically important steroid molecules as guests using the guest-responsive variation in the fluorescence emission intensity of coumarin.

Circular Dichroism↗
PubMed2003

Inhibition of peptide amyloid formation by cationic peptides with homologous sequences.

We have studied the model peptides that undergo self-initiated structural transition from alpha-helix to beta-sheet and self-assembling into amyloid fibrils. We here constructed an inhibition system of amyloid formation utilizing homologous recognition and assembly of peptides with increased solubility. Among 20 peptides with homologous sequences examined here, cationic peptides showed the stronger inhibition ability against the amyloid formation of a model peptide.

Amino Acid Sequence↗
PubMed2003

Complementary nucleobase interaction enhances peptide-peptide recognition and self-replicating catalysis.

The availability of the complementary interaction of nucleobases for influencing the formation of peptide architectures was explored. Nucleobases were incorporated as additional recognition elements in coiled-coil peptides by employing nucleobase amino acids (NBAs), which are artificial L-alpha-amino gamma-nucleobase-butyric acids. The effect of the base-pair interaction on intermolecular recognition between peptides was evaluated through a self-replication reaction. The self-replication reactions of the peptides with complementary base pairs such as thymine-adenine or guanine-cytosine at the g-g' heptad positions were accelerated in comparison with those of the peptides with mismatched base pairs or without nucleobases. However, thymine-adenine pairs at the e-e' positions did not enhance the self-replication. In the presence of a denaturant, the enhancement effects of complementary base pairs on the reaction disappeared. Thermal denaturation studies showed that the thymine-adenine pairs contributed to stabilization of the coiled-coil structure and that the pairs at the g-g' positions were more effective than those at the e-e' positions. The peptide-peptide interaction was reinforced by complementary nucleobase interactions appropriately arranged in the peptide structure; these led to acceleration of the self-replication reactions.

Amino Acid Sequence↗
PubMed2003

Novel peptides bearing pyrene and coumarin units with or without beta-cyclodextrin in their side chains exhibit intramolecular fluorescence resonance energy transfer.

Novel peptides bearing the pyrene/coumarin FRET pair in their side chains have been designed and synthesized. Peptide 1 having endogenous beta-cyclodextrin (beta-CD) in the side chain exhibits FRET in aqueous solution, indicating that coumarin, being accommodated into the CD cavity, is separated from pyrene. Guest-induced quenching of the fluorophores in 1 indicates that coumarin, being excluded from the CD cavity, comes into close contact with pyrene. Peptide 2 shows FRET only after addition of external beta-CD that again reflects the idea that beta-CD surely caps the coumarin unit in its hydrophobic cavity, and, therefore, quenching of the fluorophores can be prevented in FRET peptide probes. With this strategy, various peptide-based FRET probes can be developed that would be useful for studying biological phenomena in living cells.

Circular Dichroism↗
PubMed2003

Construction of a protein-detection system using a loop peptide library with a fluorescence label.

Construction of a novel protein-detection system was carried out using a designed peptide library with fluorescent labels based on loop structures. As a basic model study, detection of alpha-amylase using fluorescent-labeled peptides derived from an active loop of tendamistat was examined. The detection methods for proteins with immobilized peptides as well as peptides in solution have been successfully established. Based on these results, a loop peptide library that has various turn sequences grafted on a stable loop structure has been constructed. Various proteins with recognition patterns corresponding, for instance, to "protein fingerprints" could be detected using an immobilized peptide library. The present results suggest that the system can be applied to the development of a peptide microarray that behaves as a protein chip.

Amino Acid Sequence↗
PubMed2002

Amyloid architecture: complementary assembly of heterogeneous combinations of three or four peptides into amyloid fibrils.

The amyloid fibril is a misfolded and undesirable state for proteins that has been proposed to be a causative agent for a variety of fatal diseases known as amyloid diseases, such as Alzheimer's and prion diseases. However, the fibril has a highly ordered tertiary structure in which numerous beta-strand polypeptide chains align in a regular pattern. Thus, this kind of fibril has the potential to be engineered into proteinaceous materials. Amyloid fibrils of misfolded proteins primarily comprise a single polypeptide species, that is, the self-assembly is homogeneous. We here found that three or four designed peptides can assemble heterogeneously and cooperatively into amyloid fibrils, a process accompanied by a drastic secondary structural transition from alpha helix to beta sheet. Heterogeneous assembly into fibrils is accomplished by complementary electrostatic interactions between three or four peptide species, each of which is not able to self-assemble homogeneously. These findings will lead to a novel way to study the molecular details of amyloid formation and also to design beta-sheet peptidyl materials.

Amino Acid Sequence↗
PubMed2002

Nucleobase amino acids incorporated into the HIV-1 nucleocapsid protein increased the binding affinity and specificity for a hairpin RNA.

L-alpha-amino acids with a nucleobase in the side chain (nucleobase amino acids; NBAs) were used to enhance the function of RNA-binding proteins that recognize structured RNA. These NBAs were utilized in the three-dimensional structure of the protein to enhance RNA binding affinity and specificity as a result of selective recognition of NBAs by RNA bases. NBA units were incorporated at various positions into the HIV-1 nucleocapsid protein NCp7 (residues 1-55), which contains two CCHC-type (Cys-X(2)-Cys-X(4)-His-X(4)-Cys-type; X=an amino acid residue) zinc knuckle domains. The binding ability was evaluated by using the stem-loop (SL)3 region of HIV-1 Psi-RNA. Visible light absorption measurements revealed that two zinc ions bound strongly and quantitatively to the NBA-NCp7 molecule and to the wild-type NCp7 protein. This result indicates that the incorporation of NBA units composed of L-alpha-amino acids did not influence the formation of the specific structure of NCp7. Binding analysis with fluorescein-labeled SL3 RNA revealed that incorporation of NBA units into the NCp7 protein at appropriate positions increased its RNA binding affinity and specificity. An NBA-NCp7 protein that possessed cytosine and guanine NBA units at positions 13 and 46, respectively, showed a binding affinity for SL3 RNA ninefold higher than that of wild-type NCp7 as a result of the specific and cooperative interaction of the NBA units with RNA bases. These results clearly demonstrate that inclusion of NBA units in the three-dimensional structure of an RNA-binding protein is a useful strategy for enhancing the function of the protein.

Amino Acid Sequence↗
PubMed2002

Construction of the novel conformationally-restricted peptide library for screening of peptides that control the interaction between nucleobases.

A unique conformationally-restricted peptide library was constructed using a loop structure as a structural scaffold. This library was used for the screening of the amino acid sequences that control the interaction between nucleobase triplets. The peptides have PNAs at the C-terminus as the recognition site and the random amino acid sequence at the N-terminus as the effector for the interaction between PNA and its complementary DNA triplets. From the peptide libraries constructed by the positional scanning method, the sequences that affect the interaction between PNA and complementary DNA were selected. The difference in the characteristic results by using A-T and G-C pairs was presented. This study would also give us some useful information about interaction between peptides and nucleic acids, such as relevances between these biomolecules in a prebiotic era.

Animals↗
PubMedDate not supplied

De novo design of peptides with L-alpha-nucleobase amino acids and their binding properties to the P22 boxB RNA and its mutants.

A method to design novel molecules that specifically recognize a structured RNA would be a promising tool for the development of drugs or probes targeting RNA. In this study, the de novo design of the alpha-helical peptides having L-alpha-amino acids with nucleobases (nucleobase amino acids, NBAs) was carried out. Binding affinities of the peptides for a hairpin RNA derived from P22 phage were dependent on the types and positions of the NBA units they have. Some NBA peptides bound to the wild-type RNA or its mutant with high affinity and high specificity compared with the native P22 N peptide. These results indicate that the NBA units on the peptides interact with the RNA bases in a specific manner. It is demonstrated that the de novo design of peptides with the NBA units is an effective way to construct novel RNA-binding molecules.

Amino Acids↗