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H Uedaira

Publications and source records attributed to H Uedaira.

36 records · Page 2Linked to original sources
PubMed1994

Alpha-helical assembly of biologically active peptides and designed helix bundle protein.

The formation of alpha-helical assembly by complexing biologically active peptides with de novo designed protein is described. The de novo designed protein described here is a cystine-linked 4-helix bundle protein constructed with 80 amino acid residues and forms a hydrophobic core region surrounded by 4 helices in an aqueous solution. The biologically active peptides, such as melittin and human growth hormone releasing factor, contain the sequences that are able to form amphiphilic helices. These peptides alone do not form the alpha-helix structure in a diluted solution with low ion strength. But on mixing with the designed helix bundle protein, the peptides are strongly bound to the protein with the induction of alpha-helical structure in the biologically active peptides. The content of induced alpha-helix is in accord with that estimated from the amphiphilic sequence. The results mean that a novel architecture composed of alpha-helices is formed. Fluorescent and temperature-scanning measurement revealed that the alpha-helical assembly is constructed with hydrophobic interaction. Also, it is shown by means of fluorescence depolarization that the assembly has a compact globular form corresponding to 1:1 complex.

Amino Acid Sequence↗
PubMed1993

Thermal stability of the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the c-myb protooncogene product consists of three homologous tandem repeats of 51-52 amino acids (denoted as R1, R2, and R3 from the N-terminal side). In order to analyze conformational and thermodynamic characteristics of the homologous repeats, we have examined the DNA-binding domain by circular dichroism (CD) and differential scanning calorimetry (DSC). The CD spectra for the three individual repeats are significantly different in the fine profiles, indicating subtle differences in their conformations. The melting analyses for the fragments show that the thermal stability of each fragment is different from one another, with the following order of stability: R1(Tm = 61 degrees C) approximately greater than R3(57 degrees C) >> R2(43 degrees C), where R2 is much less stable than the other repeats. The denaturing process for the whole DNA-binding domain, measured by DSC, is characterized by a very broad transition ranging from 30 to 80 degrees C. The denaturation curve can be fit well by a three-state transition with one intermediate state. The transition temperature for the native-to-intermediate transition coincides with the melting temperature of R2, indicating that the intermediate state corresponds to the unfolding of unstable R2. The CD spectrum of the whole domain is almost identical to the sum of the individual spectra. Thus, these results suggest that the individual repeats in the whole DNA-binding domain behave independently in terms of conformation and stability. The addition of DNA to the DNA-binding fragment drastically changed the melting profile, in which the broad transition curve was replaced by a sharp peak at 58 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Diminution of biological reactivity of ethanol by changing the solution structure by weak ultrasonication.

The weak ultrasonication (40 kHz, 12 mW, 1 week) of ethanol solutions was found to reduce stimulation of the senses of smell and taste by the ethanol on the basis of blind tests with an aqueous ethanol solution (33.0% w/v) and an immature distilled spirit (25.0% v/v). Experiments on mice also demonstrated that a treated aqueous ethanol solution had a weaker depressant effect on the central nervous system, as evaluated by the relative frequency with which mice regained the righting reflex at a dose of either 4.0 or 4.5 g/kg (p < 0.05 or p < 0.01, respectively) and by the reduction in rectal temperature at a dose of 5.0 g/kg (p < 0.05) soon after ethanol administration. Analyses of both the ethanol concentration by head-space gas chromatography and the free radicals by electron spin resonance spectrometer failed to reveal any chemical changes in aqueous ethanol solutions subjected to weak ultrasonication. However, measurement of the spin-lattice relaxation time (T1) of the 2H of water molecules by 2H-NMR showed that the treatment slightly accelerated the thermal motion of water molecules in the solutions. Treated solutions were also found to have a slightly higher density than untreated ones. These physical data demonstrate that weak ultrasonication induces a structural change, such as a more compact and homogeneous structure by changing the microdynamic behavior of the solution. These biological and physical studies suggest that only a slight structural change in an ethanol solution induces a marked change in the biological reactivity of ethanol without any chemical modification of the solution itself.

Adult↗
PubMed1993

1H-NMR spectroscopic analysis of human urine: periodic variation of trimethylamine oxide excretion.

We used 1H-NMR to study the constituents of the urine from a female volunteer who had an unidentified complaint syndrome (chief complaints: discomfort of scapular region, abdominal distention and exhaustion). The pH dependence of the chemical shift of some constituents was investigated for signal assignments, and the intense peak at about 3.3 ppm was assigned to trimethylamine oxide (TMAO). We measured the concentration of the constituents of urine, and TMAO excretion in the urine over a three week time period. The TMAO excretion fluctuated in a cycle of about 2 days. The average excretion determined (3.8 mmole/day) is considerably higher than data of healthy humans that had been previously reported (0.3 to 0.5 mmole/day).

Adult↗
PubMed1991

Fluctuation and rotation of human growth hormone-releasing factor in the presence and the absence of phospholipid bilayer analyzed by time-resolved fluorescence depolarization.

Time-resolved fluorescence depolarization measurements were carried out for human growth hormone-releasing factor analog ([Trp10]-hGRF (1-29) NH2), where the Trp10 residue was incorporated as a fluorescent probe, in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phospho-rac- glycerol(DMPG) liposome and in aqueous 2,2,2-trifluoroethanol (TFE) solution. The fluorescence lifetimes and the rotatory correlation times of the peptide in each medium were determined. The apparent volumes of the rotatory Brownian motion unit calculated from these fluorescent parameters indicate the different mode of the fluctuation and/or the rotation of the peptide in each medium, such as: (i) In the aqueous solution, several segments of the peptide fluctuate individually. (ii) In the DMPG bilayer, both the local fluctuation of Trp residue alone and the rotation of the whole molecule exist. (iii) In the aqueous TFE solution, the monomeric peptide rotates as a rigid ellipsoid.

Amino Acid Sequence↗
PubMed1991

Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane.

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.

Amino Acid Sequence↗
PubMed1991

Asymmetric inclusion by de novo designed proteins: fluorescence probe studies on amphiphilic alpha-helix bundles.

The inclusion feature and supersecondary structure of the de novo designed proteins which are constructed with several amphiphilic alpha-helices and flexible linkage parts were investigated with fluorescence probes. Five types of small proteins (or peptides) have been designed, which are composed of 2, 3, 4, 4, and 6 helices, respectively, and are linked with only linear junctions except for one of 4-helix proteins. All of these proteins have inclusion ability for hydrophobic fluorophores. Further, by the analysis of fluorescence polarization anisotropy, it was suggested that these proteins include guest molecules in compact helix bundles constructed with about 4 helices. Asymmetric inclusion of both monomer and stacked dimer of acridine orange derivatives was found by means of induced circular dichroism except for the 4-helix protein with cross-junction. The chirality of the included dimer proved to be in accordance with the chiral sense of alpha-helical coiled-coil. The 6-helix protein has especially high efficiency in inclusion for any fluorophores examined in this study and brings about a significant blue-shift of maximal emission for 8-anilino-1-naphthalenesulfonate.

Acridine Orange↗
PubMed1991

Hydration of clupeine in solution.

Spin-lattice relaxation times, T1, of H2(17) O at 25 degrees were measured for aqueous solutions of clupeine and its constituent amino acids, which are serine, threonine, proline and arginine. The dynamic hydration numbers, nDHN, of clupeine and amino acids were determined from a concentration dependence of T1. The coordination numbers nh, and the rotational correlation times, tau ch, of water molecules around clupeine and amino acids were estimated and compared with that of pure water. The tau ch/tau co of clupeine was 1.85 and close to that of arginine. The experimental value of nDHN of clupeine was in good agreement with that calculated from the nDHN values of the constituent amino acids. This means that the clupeine molecule has a random conformation in solution.

Amino Acid Sequence↗
PubMed1990

Structural organization of flagellin.

The terminal regions of flagellin from Salmonella typhimurium have been reported to be disordered in solution, whereas the central part of the molecule contains protease-resistant, compact structural units. Here, conformational properties of flagellin and its proteolytic fragments were investigated and compared to characterize the domain organization and secondary structure of flagellin. Deconvolution analysis of the calorimetric melting profiles of flagellin and its fragments suggests that flagellin is composed of three co-operative units or domains. The central part of the molecule, residues 179 to 418, consists of two domains (G1 and G2), whereas the third domain (G3) is discontinuous, constructed from segments 67 to 178 and 419 to 448. Secondary structure prediction and analysis of far-ultraviolet circular dichroic spectra have revealed that G1 and G2 consist predominantly of beta-structure with a little alpha-helical content. G3 contains almost equal amounts of alpha and beta-structure, while in the terminal parts of flagellin the ordered secondary structure seems to be entirely alpha-helical.

Bacterial Proteins↗
PubMed1990

Thermal stability of dihydrofolate reductase and its fused proteins with oligopeptides.

Two fused proteins of dihydrofolate reductase (DHFR) with oligopeptides were prepared by a recombinant DNA method. One of these, DHFR-IQI, has three (Ile-Gln-Ile) and the other, DHFR-lek, has eight (Ile-Arg-Met-Tyr-Gly-Gly-Phe-Leu) additional amino acid residues at the C terminals; in both proteins, Cys152 of wild DHFR is replaced by Glu. The thermal transition of the proteins was measured by CD and DSC at pH 7.0 and compared with that of wild DHFR. The results show that the thermal stability of DHFR-IQI is the same as that of the wild DHFR and that of DHFR-lek is less than that of the former two DHFRs. Analysis of the DSC data of DHFR-IQI indicates that the thermal transition is a three-state one. Data from both DSC and CD measurements suggest the association of DHFR-lek molecules.

Calorimetry, Differential Scanning↗
PubMed1989

Acoustical investigation of poly(dA).poly(dT), poly[d(A-T)], poly(A).poly(U) and DNA hydration in dilute aqueous solutions.

Apparent molar adiabatic compressibilities and apparent molar volumes of poly[d(A-T)].poly[d(A-T)], poly(dA).poly(dT), DNA and poly(A).poly(U) in aqueous solutions were determined at 1 degree C. The change of concentration increment of the ultrasonic velocity upon replacing counter ion Cs+ by the Mg2+ ion was also determined for these polymers. The following conclusions have been made: (1) the hydration of the double helix of poly(dA).poly(dT) is remarkably larger than that of other polynucleotides; (2) the hydration of the AT pair in the B-form DNA is larger than that of the GC pair; (3) the substitution of Cs+ for Mg2+ ions as counter ions results in a decrease of hydration of the system polynucleotide plus Mg2+, and (4) the magnitude of this dehydration depends on the nucleotide sequence; the following rule is true: the lesser is a polynucleotide hydration, the larger dehydration upon changing Cs+ for Mg2+ ions in the ionic atmosphere of polynucleotide.

Base Composition↗
PubMed1979

Na+ interacting with gramicidin D. A nuclear magnetic resonance study.

23Na nuclei in a milk-white emulsion composed to nonionic surfactant and higher alcohol in saline were characterized by single values of T1 and T2 and a single Larmor frequency. In the presence of small amounts of gramicidin D (Dubos), the relaxations of 23Na were greatly accelerated, and the transverse relaxation was a sum of two decaying exponentials. But only a single T1 was observed; it was roughly equal to the slow T2. The slow T2 accounted for about 40% of the total resonance intensity. The relaxation rates increased linearly with the increase of the gramicidin concentration. The absorption signal consisted of a narrow and a broad line, both centered at the same frequency. The present results suggest that nuclear magnetic resonance spectroscopy is a useful tool for studying the nature of ion-permeable channels of biological membranes, even when the channel has no ionizable groups.

Gramicidin↗
PubMedDate not supplied

Thermodynamic databases for proteins and protein-nucleic acid interactions.

Thermodynamic data regarding proteins and their interactions are important for understanding the mechanisms of protein folding, protein stability, and molecular recognition. Although there are several structural databases available for proteins and their complexes with other molecules, databases for experimental thermodynamic data on protein stability and interactions are rather scarce. Thus, we have developed two electronically accessible thermodynamic databases. ProTherm, Thermodynamic Database for Proteins and Mutants, contains numerical data of several thermodynamic parameters of protein stability, experimental methods and conditions, along with structural, functional, and literature information. ProNIT, Thermodynamic Database for Protein-Nucleic Acid Interactions, contains thermodynamic data for protein-nucleic acid binding, experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data have been incorporated into 3DinSight, an integrated database for structure, function, and properties of biomolecules. A WWW interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. These thermodynamic databases, together with structural databases, help researchers gain insight into the relationship among structure, function, and thermodynamics of proteins and their interactions, and will become useful resources for studying proteins in the postgenomic era.

DNA↗