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Y Kyogoku

Publications and source records attributed to Y Kyogoku.

At least 55 records · Page 3Linked to original sources
PubMed1996

Analysis of substrate specificity of the RuvC holliday junction resolvase with synthetic Holliday junctions.

The Escherichia coli RuvC protein endonucleolytically resolves Holliday junctions, which are formed as intermediates during genetic recombination and recombination repair. Previous studies using model Holliday junctions suggested that a certain size of central core of homology and a specific sequence in the junction were required for efficient cleavage by RuvC, although not for binding. To determine the minimum length of sequence homology required for RuvC cleavage, we made a series of synthetic Holliday junctions with various lengths of homologous sequence in the core region. It was demonstrated that a monomobile junction possessing only 2 base pairs of the homology core was efficiently cleaved by RuvC. To study the sequence specificity for cleavage, we made 16 bimobile junctions, which differed only in the homologous core sequence. Among them, 6 junctions were efficiently cleaved. Cleavage occurred by introduction of nicks symmetrically at the 3'-side of thymine in all cases. However, the nucleotide bases at the 3'-side of the thymines were not always identical between the two strands nicked. These results suggest that RuvC recognizes mainly topological symmetry of the Holliday junction but not the sequence symmetry per se, that the thymine residue at the cleavage site plays an important role for RuvC-mediated resolution, and that a long homologous core sequence is not essential for cleavage.

Bacterial Proteins↗
PubMed1996

Solution Strucutres of DNA duplexes containing a DNA x RNA hybrid region, d(GG)r(AGAU)d(GAC) x d(GTCATCTCC) and d(GGAGA)r(UGAC) x d(GTCATCTCC).

The solution structures of two DNA duplexes containing a DNA*RNA hybrid region at different sites, d(GG)r(AGAU)d(GAC) x d(GTCATCTCC) (DHD, where D and H represent the DNA and DNA x RNA hybrid segments, respectively) and d(GGAGA)r(UGAC) x d(GTCATCTCC) (DDH), were determined by nuclear magnetic resonance spectroscopy to clarify the structural features of the D-H and H-D junctions. All proton-proton distances were derived from the NOESY spectra, with mixing times of 45 ms, and the restrained molecular dynamics were carried out starting from the typical A- and B-form conformations. Both duplexes were converged from the respective initial structures into structures with RMSD values of less than 1.0 angstrom. These structures were subjected to full relaxation matrix refinement to produce the final structures. In the case of the D-H junction, where the ribonucleotide was linked to the 3'-end of the DNA, the H2' and H2" signals of the deoxynucleotide overlapped completely, and the ribonucleotide had a H1'-H2' coupling constant larger than that of the normal C3'-endo sugar pucker. The dihedral angles, the pseudorotation phase angles, and the helical parameters changed at the H-D junction, but not at the D-H junction. A detailed comparison of the two duplexes revealed the structural heterogeneity between the DNA segment and the DNA x RNA hybrid region and the transitions at the junctions.

Base Sequence↗
PubMed1996

Regulation of the redox order of four hemes by pH in cytochrome c3 from D. vulgaris Miyazaki F.

The assignment of 1H-NMR signals of the heme methyl and propionate groups of cytochrome c3 of D. vulgaris Miyazaki F was performed. The heme assignment was revised for hemes 2 and 3 (sequential heme numbering). Namely, heme 4 is mainly reduced at first with hemes 1, 2 and 3 following it in this order. The p2H titration of heme methyl signals in four macroscopic oxidation states was performed in the p2H range of 5.2 to 9.0. While the heme methyl resonances in the fully oxidized state showed just small changes with p2H, most resonances in the intermediate oxidation states revealed clear p2H dependence. In particular, the methyl resonances of heme 1 shifted significantly in the acidic region. Then, the chemical shifts of beta-CH2 (next to the carboxyl group) of all propionate groups in the fully oxidized state were observed at various p2H in the range of 4.5 to 9.0. Only the propionate group at C-13 (IUPAC-IUB nomenclature) of heme 1 showed a clear change in this p2H range, its titration curve being similar to those of the methyl resonances of heme 1 in the intermediate oxidation states. pKa of the propionate group was 5.95 +/- 0.05. Analysis of the microscopic formal redox potentials was carried out for the observations at p2H 5.2, 7.1 and 9.0. The redox potentials of heme 1 showed the most remarkable p2H dependence, resulting in the change of the order of the redox potentials of four hemes. A significant change was also found in the interacting potential between hemes 1 and 2. In the light of the p2H-titration experiments, the propionate at C-13 of heme 1 was identified as the most plausible ionizable group responsible for the p2H dependence of microscopic redox potentials of heme 1 in the acidic region.

Cytochrome c Group↗
PubMed1996

15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1-24).

For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required. A direct over-expression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.

Amino Acid Sequence↗
PubMed1996

Characterization and comparison of synthetic immobile and mobile Holliday junctions.

Eight synthetic Holliday junction (HJ) oligonucleotides containing an immobile or a mobile junction were characterized by gel electrophoresis, ultraviolet absorption and circular dichroism (CD) spectroscopy. Four 24-mer deoxyribonucleotides formed stable immobile and mobile HJs in 0.1 M NaCl at 5 muM strand concentration at room temperature. However, the immobile HJ constructed from four 18-mers was less stable, and four 12-mers did not form the HJ structure under the conditions used. A comparison of the melting profiles of the HJs with those of the duplexes corresponding to the arms of four-way junctions indicated that the thermal stability of the HJ was similar to that of the individual arm and the cooperativity of the melting behavior of the HJ was relatively higher than that of the individual arm duplex. The Tms of the mobile HJs containing 4, 6, 8, and 10 base-pair homologous cores at junctions were essentially identical with that of the immobile HJ of the same size. There is a tendency that the HJ containing a larger homologous core region becomes more resistant to thermal denaturation. The addition of divalent metal cations, Mg2+ and Ca2+, to the solutions of the HJs raised their melting temperatures. The difference found for the CD spectra of the HJs which differ only in the arrangement of the HJ depended primarily upon the DNA sequence flanking the junction. The RuvC protein binds to the immobile and mobile HJs, regardless of the presence and the size of the homologous core at the junction.

Bacterial Proteins↗
PubMed1995

Three-dimensional dimer structure of the lambda-Cro repressor in solution as determined by heteronuclear multidimensional NMR.

The 1H, 15N and 13C magnetic resonances of the lambda-Cro repressor have been assigned almost completely, mainly through the use of heteronuclear multidimensional NMR methods. Inter-subunit NOEs were distinguished by means of heteronuclear spectral editing technique (13C double half filter technique). Based on the distance and dihedral angle constraints derived from the NMR data, the three-dimensional solution structure of the lambda-Cro repressor in the dimeric form has been calculated by the simulated annealing method. The input for the structure calculations consisted of 1H-1H distance constraints, of which 1536 were intra-subunit and 40 were inter-subunit, and dihedral angle, phi, constraints, which numbered 92. The average root-mean-square deviation (RMSD) for all backbone heavy- atoms of the 20 calculated structures for residues 3 to 59 of the total of 66 amino acid residues in both subunits was 1.57 Angstrum, while the average RMSD for each subunit in the same residue range was 0.66 Angstrum. The subunit is composed of three alpha-helices, residues 7 to 13, 16 to 23 and 27 to 36, and a three-stranded anti-parallel beta-sheet composed of residues 3 to 6, 40 to 44 and 50 to 55. The solution structure of the subunit is essentially the same as that in the crystalline form, but the structure of the dimer form in solution differs from that of the dimer unit in the crystalline form. It is suggested that the solution dimer structure is distorted to fit the recognition helices in the major grooves of DNA on complex formation.

Amino Acid Sequence↗
PubMed1995

Solution structure of the activator contact domain of the RNA polymerase alpha subunit.

The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alpha CTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop. Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

Base Sequence↗
PubMed1995

Solution structure of omega-conotoxin MVIIC determined by NMR.

The solution structure of the P- and Q-type Ca2+ channel blocker, omega-conotoxin MVIIC (a peptidic neurotoxin composed of 26 amino acid residues), has been determined by 1H-NMR and simulated annealing calculations. The resulting calculated structures converged very well to a conformation with an average value of pairwised RMSD for N, C alpha and C' of 0.62 A. Lys-25 is buried in the molecule and less flexible so that among the four Lys residues, its side chain provides the lowest reactivity on biotinylation and the mono-biotinylation in this residue less influences the biological activity.

Amino Acid Sequence↗
PubMed1995

Secondary structure and folding topology of the DNA binding domain of interferon regulatory factor 2, as revealed by NMR spectroscopy.

The secondary structure elements of the DNA-binding domain of mouse interferon regulatory factor 2 [IRF-2(113)] were determined by heteronuclear multidimensional NMR spectroscopy. The sequential NOE connectivities, amide proton exchange rates, and 3JHN alpha coupling constants indicated the presence of three alpha-helical regions and four short beta-strands connected through relatively long loops. The long range NOEs indicated the four strands form an antiparallel beta-sheet and the three alpha-helices form a bundle on the sheet. The arrangement of the secondary structure elements and the overall folding topology resemble those of the DNA binding domains of bacterial activator CAP, heat shock transcription factors, and fork-head proteins, although there is no sequence homology among them.

Amino Acid Sequence↗
PubMed1995

Structure of the Oct-3 POU-homeodomain in solution, as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.

The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed.

Amino Acid Sequence↗
PubMed1995

Nuclear magnetic resonance study of complexes between CRP and its 22- and 28-base-pair operators.

The binding of the cAMP receptor protein (CRP) to the portion of the lac promoter comprising the core of the CRP recognition sequence has been investigated. The effect of the binding of CRP to the symmetrical 22- and 28-base-pair operators was investigated by 1H NMR. The binding of cAMP*CRP to the 22mer DNA did not bring about any changes in the chemical shift values of the imino proton resonances of the DNA, but did cause selective line broadening of the imino proton resonances of specific base pairs (TA 4, GC 5). These base pairs are contained in the motif 5'(TGTGA)3', which is thought to be the region crucial to interaction with the CRP. The binding of cAMP*CRP to the 28mer DNA brought about large changes in the imino proton resonances that seem to be induced by DNA bending. Therefore it seems likely that CRP requires DNA longer than a 22mer to bend it. We also used a C-terminal protease-digested CRP (CRPcy) in a DNA-binding experiment. The binding of cAMP*CRPcy to the 28mer DNA did not induce bending. These results indicate that the C-terminal region of CRP participates in DNA binding and is important for DNA bending.

Base Composition↗
PubMed1995

Solution structure of a human calcitonin analog elucidated by NMR and distance geometry calculations.

Three-dimensional structure of a human calcitonin analog (abbreviated as hCTa) in which the amino acids of the wild type are replaced at position 12, 16 and 19 by leucine residues and further at position 22 by a tyrosine residue was studied in TFE solution by 1H-NMR and distance geometry calculations. This analog has a 15-20 times activity as compared with the wild type. The amino acid replacements resulted in formation of an amphiphilic alpha-helix in the region between the residues 4-20. The overall three-dimensional structure is similar to that of the wild type. The conformational feature of hCTa with a hydrophobic face composed with a Met and four Leu residues may be related to its higher hypocalcemic potency.

Amino Acid Sequence↗
PubMed1995

Isolation of a cold-sensitive fermentation mutant of a baker's yeast strain and its use in a refrigerated dough process.

Conventional baker's yeast converts sugars in dough into CO2 and ethanol to a significant extent when the dough is stored for days at 5 degrees C. We have isolated Csf (cold-sensitive fermentation) mutants of a commercial baker's yeast by a selection method including as the critical step a nystatin treatment to mutagenized cells at 10 degrees C in the presence of antimycin A. The fermentative activity of mutant strain CSF3 was substantially zero at 5 degrees C and one-fifth that of the parent at 10 degrees C but was restored to the same level as the parental activity at 25 to 40 degrees C. In contrast with the parent, the mutant strain normally produced white bread dough and butter roll dough even after the dough was stored for a week at 5 degrees C.

Antimycin A↗
PubMed1995

Structural requirement of four-way junction DNA for binding to and cleavage by RuvC.

Sixteen bimobile four-way junction DNAs were designed to clarify the consensus sequence at the resolution site by the E. coli RuvC protein. The consensus sequence is 5'-T decreases X-3' (X: A, G, C, and T) without exception, but the sequences at the cleaved sites are not necessarily symmetric. The presence of symmetrically located two AT base-pairs at the junction is the minimal requirement for resolution.

Bacterial Proteins↗
PubMed1995

Topology of triple helical DNA.

From the stereochemical modeling of a triple helix, the third poly(dT) strand can bind in anti-parallel direction to the poly(dA) strand from the major groove of the poly(dA)-poly(dT) double helix. We designed a single strand 46mer DNA named AP46 which may form an anti-parallel triple helix containing only ATT base triplet by folding back in the strand. The UV melting profile of AP46 showed an increase of absorbance at 260 nm as temperature was raised, but no obvious double transition points were observed. We examined the type of a triple helix by using 3'-5' exonuclease activity of T4 DNA polymerase. The digestion experiment indicates that an anti-parallel triple helix is not so stable even if it exists. This enzymatic method is useful for probing the secondary structure of DNA strand.

Base Sequence↗
PubMed1994

Conformational analysis of elcatonin in solution.

The conformational characteristics of elcatonin, an analogue of eel calcitonin having a disulfide bond Cys1-Cys7 replaced by an ethylene linkage between residues 1 and 7, have been analyzed in aqueous trifluoroethanol solutions. Circular dichroic spectra of elcatonin and eel calcitonin itself reveal the presence of alpha-helices at trifluoroethanol concentrations above 15%. The spectral changes caused by the trifluoroethanol content of the solutions are interesting. An isosbetic point is detected for eel calcitonin indicating that a conformational transition occurs between two states, namely alpha-helical and random coil states. On the other hand, the CD curves of elcatonin at less than 15% trifluoroethanol deviate from the isosbetic point while those at higher concentration are similar to those of eCT. This can probably be attributed to the third element of the ordered structure of elcatonin which is formed in 15% trifluoroethanol. The solution conformation of elcatonin in a mixture of 60% water and 40% trifluoroethanol has been determined by the combined use of 1H-NMR spectroscopy and distance geometry calculations. The conformation is characterized by an amphiphilic alpha-helix between Thr6 and Thr21, which extends into the constrained cyclic portion of the molecule to Thr6. The third structural element of elcatonin found in the CD analysis is detected by some turn structures in the region between residue 1 and Ser5 in the calculated structure.

Amino Acid Sequence↗