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Biomedical subjects

Y Kyogoku

Publications and source records attributed to Y Kyogoku.

At least 73 records · Page 4Linked to original sources
PubMed1994

Nuclear magnetic resonance study of the interaction of T4 endonuclease V with DNA.

T4 endonuclease V catalyzes the DNA strand cleavage in the vicinity of a thymine dimer. In order to obtain insight into the specific recognition mechanism of this enzyme with a thymine photodimer within DNA, the conformations of five different DNA duplexes, [sequence: see text] with which the enzyme can interact, were studied by 1H NMR. DNA I, DNA IV, and DNA V do not contain the TT sequence or a thymine dimer and hence, are expected to bind the enzyme only in a nonspecific manner. DNA II includes a single TT sequence which does not form a thymine dimer. Only DNA III is expected to bind specifically to the enzyme through a thymine photodimer. The NMR spectra of these five DNA duplexes in the absence of the enzyme clearly show that the formation of a thymine dimer within the DNA induces only a minor distortion in the structure and that the overall structure of B-type DNA is retained. The photodimer formation is found to cause a large change in chemical shifts at the GC7 base pair, which is located at the 3'-side of the thymine dimer, accompanied by the major conformational change at the thymine dimer site. The effects of T4 endonuclease V binding on these DNA duplexes were also investigated by 1H NMR. The binding of this enzyme to DNA I, DNA IV, and DNA V causes no alteration in chemical shift values of the imino proton resonances, but the binding to DNA II induces a small downfield shift in the imino proton resonance of GC7.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1994

Filtering methods for selection of singlet and doublet signals in NMR spectra of DNA oligomers.

The base proton (purine H8 and pyrimidine H6) resonances are key signals for the assignment of the proton resonances of DNA oligomers. They are classified into two groups, i.e., cytosine H6 signals, observed as doublets, and the other base proton signals, observed as singlets. Here we propose some experiments for distinguishing the cytosine H6 signals from the other base proton signals. Moreover, the ability of signal selection and the sensitivity as to signal detection were compared for all experiments, and the optimum conditions for spectral measurements were surveyed. Some of the experiments were employed as the NOESY detection pulse. Previously proposed experiments, such as HOENOE and HAL, were also used in the comparison.

Cytosine↗
PubMed1994

Dynamic structures of intact chicken erythrocyte chromatins as studied by 1H-31P cross-polarization NMR.

The dynamic properties of DNA in intact chicken erythrocyte cells, nuclei, nondigested chromatins, digested soluble chromatins, H1, H5-depleted soluble chromatins and nucleosome cores were investigated by means of single-pulse and 1H-31P cross-polarization NMR. The temperature dependence of the phosphorus chemical shift anisotropy was identical for the former three in the presence of 3 mM MgCl2, suggesting that the local higher order structure is identical for these chromatins. The intrinsic phosphorus chemical shift anisotropy of the nucleosome cores was -159 ppm. The chemical shift anisotropy of DNA in the chromatins can be further averaged by the motion of the linker DNA. The spin-lattice relaxation time in the rotating frame of the proton spins (T1p) of the nondigested chromatins was measured at various locking fields. The result was analyzed on the assumption of the isotropic motion to get a rough value of the correlation time of the motion efficient for the relaxation, which was eventually ascribed to the segmental motion of the linker DNA with restricted amplitude. The 30 nm filament structure induced by NaCl was shown to be dynamically different from that induced by MgCl2. Side-by-side compaction of 30-nm filaments was suggested to be induced in the MgCl2 concentration range higher than 0.3 mM. Biological significance of the dynamic structure was discussed in connection with the results obtained.

Animals↗
PubMed1994

Dynamic properties of nucleic acids in biosupramolecular systems, as studied by 31P NMR.

The dynamic properties of nucleic acids in five different types of intact supramolecular systems, namely, chicken erythrocyte chromatin, the wild type and a deletion mutant of the lambda phage, lipid-containing phage PM2, and Alteromonas espejiana ribosomes, were investigated by means of 31P solid-state NMR. The nucleic acids in the different supramolecular systems showed unique dynamic properties, which are closely connected with their functions. The total anisotropy of the phosphorus chemical shift (delta sigma = sigma 33-sigma 11) of the ribosomes was 210 ppm at 5 degrees C. This anisotropy was much larger than those of any DNA complexes, suggesting the highly rigid structure of ribosomal RNA. In contrast, 160 ppm was the largest chemical shift anisotropy at 5 degrees C for B-form DNA in the supramolecular systems. This flexibility would be essential for DNAs to exert their functions. The involvement of a condensation protein in the PM2 phage was supported by the chemical shift anisotropy. The spin-lattice relaxation time in the proton rotating frame [T1 rho(H)] of the nucleic acids became shorter with the increase in the effective field in the rotating frame for all systems examined, showing that the motions of the nucleic acids effective for the relaxation are in the slow motional regime or in the range of omega 1 tau c = 1 at 5 degrees C. The motional state of DNA of the lambda phage was found to change at about 20 degrees C on the basis of the temperature dependence of the spin-lattice relaxation time of phosphorus (T1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

AP-811, a novel ANP-C receptor selective agonist.

AP-811 is a derivative of the Phe8-Ile15 region of atrial natriuretic peptide (ANP) and is one of the smallest linear ligands for ANP receptors. The binding and agonist activities of AP-811 have been compared with those of other ANP analogs for the ANP-A and ANP-C receptors. AP-811 binds with a high binding affinity to and is a strong agonist for the ANP-C receptor, indicating that the binding and agonist sites for this receptor are the same or near each other in the ANP sequence. In contrast, AP-811 showed no agonistic effect for the ANP-A receptor, although it could bind to this receptor. Comparing the biological activities of AP-811 with those of other ANP analogs, we propose that the binding and agonist sites for the ANP-A receptor may consist of separate regions of ANP. In conclusion, AP-811 is the smallest C-receptor-selective agonist.

Adenylyl Cyclase Inhibitors↗
PubMed1993

Nuclear magnetic resonance study of the codon-anticodon interaction in Bombyx mori tRNA(GCCGly).

NMR spectra of Bombyx mori tRNA(GCCGly) were recorded in the GCC absence and presence of the oligonucleotide, GGCUp, which contains the codon sequence, GGC. The difference between the spectra with and without the codon oligonucleotide indicates the appearance of five new imino proton peaks. For the assignment of these peaks, G*GCUp, in which the 5'-terminal G was enriched with 95% 15N, was prepared (G*, 15N-labeled guanosine). In the imino proton spectrum of B. mori tRNA(GCCGly) on the addition of G*GCUp, the peak at 12 ppm became a doublet due to coupling with 15N nuclei. In the two-dimensional 1H-15N heteronuclear multiple-quantum correlation (HMQC) spectrum, only the peak at 12 ppm was observed, and thus it was assigned to the imino proton of the 5'-terminal G of GGCUp interacting with tRNA(GCCGly). Judging from the temperature effect and chemical shifts, the five new imino proton peaks are presumed to be due to three G.C base pairs, induced by the codon-anticodon interaction, and one U.U base pair, induced by an interaction between the 3' terminal U of GGCUp and U33 neighboring the anticodon. The binding of three trinucleotides (GGCp, GGUp and GCUp) to B. mori tRNA(GCCGly) was also investigated. Ultracentrifugation analysis showed that tRNA(GCCGly) underwent dimerization through the anticodon-anticodon interaction, but the dimerization was broken on addition of GGCUp. On 1H-NMR and ultracentrifugation analysis, it was found that GCUp not complementary to the anticodon also binds to the anticodon loop.

Animals↗
PubMed1993

Secondary structure of the oct-3 POU homeodomain as determined by 1H-15N NMR spectroscopy.

Most of the 1H and 15N magnetic resonances of the 66 amino acid long POU homeodomain of mouse Oct-3 have been assigned by the combined use of the two-dimensional homonuclear, and two- and three-dimensional heteronuclear NMR methods. The sequential NOE connectivities and amide proton exchange measurements indicate the presence of three helical regions within the domain. The positions of the three helices correspond well to those of other homeodomains, the three-dimensional structures of which have already been determined. The present NMR study provides the first experimental evidence for the existence of a helix-turn-helix motif in the oct-3 POU homeodomain.

Amino Acid Sequence↗
PubMed1993

Assignment of 1H, 15N, and 13C resonances, identification of elements of secondary structure and determination of the global fold of the DNA-binding domain of GAL4.

Almost complete assignments of the 1H, 15N, and aliphatic 13C resonances of the 62-residue N-terminal DNA-binding domain of GAL4 [GAL4(62)] have been obtained using a combination of two-dimensional homonuclear and two- and three-dimensional double- and triple-resonance heteronuclear NMR methods. The sequential NOE connectivities, amide proton exchange measurements, and 13C alpha chemical shift data indicate the presence of two short alpha-helices in the N-terminal half of the polypeptide. Residues 1-9 and 41-62 appear to be unstructured and flexible in solution. Analysis of the 13C alpha chemical shifts also revealed a significant downfield shift of approximately +3 ppm, relative to random-coil values, for the four nonbridging Zn(II) ligands, Cys 14, 21, 31, and 38. Interestingly, no such correlation was observed for the two bridging ligands, Cys 11 and 28. Preliminary structure calculations using a subset of distance restraints derived from three-dimensional 1H-15N and 1H-13C NOESY-HSQC spectra are consistent with the recently reported solution structures of Zn(II)2GAL4(7-49) [Kraulis, P., et al. (1992) Nature 356, 448-450] and of Cd(II)2GAL4(65) [Baleja, J. D., et al. (1992) Nature 356, 450-453].

Amides↗
PubMed1993

Crystallographic characterization of a PHO4-DNA complex.

Crystals have been obtained of the DNA-binding domain of the yeast transcription factor PHO4 in complexes with several synthetic fragments of DNA with appropriate cognate sequences. Crystals suitable for X-ray diffraction studies were produced in the case of a complex of the protein with a 17 base-pair fragment of DNA from a solution in polyethylene glycol and calcium chloride. The crystals have the space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 56.7 A, c = 447.8 A. The diffraction data at 3 A resolution were collected using synchrotron radiation with a Weissenberg camera for macromolecular crystallography.

Base Sequence↗
PubMed1993

Characterization of the DNA binding domain of the mouse IRF-2 protein.

The DNA binding domain of the interferon regulatory factor-2 protein (IRF-2) has been produced and characterized. alpha-chymotrypsin digestion of the purified IRF-2 protein bound to a synthetic binding site yields a peptide fragment of 14 K in molecular weight. N-terminal analysis of this peptide fragment showed that its sequence is the same as that of the intact IRF-2. A peptide fragment of approximately 14 K, IRF-2(113), which corresponds to the N-terminal 113 amino acids of the intact IRF-2 protein, has been expressed in a functional form in Escherichia coli. The first methionine was processed during the expression and the purified IRF-2(113) thus contains 112 amino acids. DNase I footprinting and gel retardation assaying showed that IRF-2(113) binds to a synthetic DNA having the consensus binding site and to the upstream regulatory sequence of the IFN-beta gene as intact IRF-2 does. These results showed that this peptide fragment, IRF-2(113), may be a good material for investigation of the DNA binding domain of IRF-2 and of the DNA-protein interaction.

Amino Acid Sequence↗
PubMed1993

Three-dimensional structure of human insulin-like growth factor-I (IGF-I) determined by 1H-NMR and distance geometry.

The three-dimensional structure of human insulin-like growth factor-I has been determined through a combination of NMR measurements and distance geometry calculations. A total of 320 interatomic distance constraints, including 12 related to the disulfide bridges, were used in these calculations. The resulting structure is characterized by the presence of three helical rods corresponding to the sequence regions, Ala8-Cys18, Gly42-Cys48 and Leu54-Cys61. Furthermore, a turn structure and an extended structure exist in the Gly19-Gly22 and Phe23-Asn26 regions, respectively. Neglecting the N- and C-termini, with their expectedly high degree of mobility as well as a fluctuating C-domain, the r.m.s.d. value is 1.9 A for backbone atoms. Those of the three alpha-helical regions are 1.0, 0.9 and 0.8 A, respectively, 1.8 A being that for the total backbone atoms participating in the formation of these three helices, showing the good convergence of their spatial arrangements. The overall structure obtained here shows that the human IGF-I molecule folds into a spatial structure very similar to that of insulin in an aqueous solution.

Amides↗
PubMed1993

Structural analyses of synthetic immobile Holliday junction.

In order to study the structural properties of Holliday junctions, we designed and synthesized oligodeoxyribonucleotides to construct the models of the Holliday junctions with no sequence symmetry at the sites of branching called immobile junctions. Electrophoretic and spectroscopic analyses of the immobile junctions indicated that the Holliday junctions constructed from four deoxy 24-mers and four 18-mers were stable in 0.1 M NaCl at approximately 5 x 10(-6) M strand concentration at room temperature. Addition of MgCl2 to the solution of HJs resulted significantly in an increase in melting temperature of HJs.

Bacterial Proteins↗
PubMed1993

Precise analyses of DNA structure by NMR.

Novel 1H NMR techniques were developed and applied to the analyses of the DNA structure. They distinguished the base proton signals of cytosine (uracil) from those of the other bases. Two dimensional experiments were also performed by using these techniques, and were found to be useful for signal assignments. Moreover, the (2'R)-[2'-2H]-labeled DNA 10-mer and 17-mer were synthesized to be used for the determination of precise structures. These stereoselective [2'-2H]-labeling made possible explicit stereospecific assignments and exact determination of the vicinal coupling constants, 3JHH, and thus the conformation of each deoxyribose ring was accurately determined.

Cytosine↗
PubMed1992

Determination of the DNA binding site of the GAL4 protein. A photo-CIDNP study.

After the assignment of 1H NMR signals of aromatic side chains by means of specific deuteration, we analyzed the DNA binding site of GAL4 by measuring photo-CIDNP spectra. The results showed that Trp36 is involved in both the specific interaction with UASG and non-specific DNA binding. This residue is located inside the Cys-rich region, but outside the putative Zn-finger. The photo-CIDNP spectrum also showed that the side chains of Tyr40 and His53 are not exposed on the surface of the protein.

Amino Acid Sequence↗
PubMed1992

An oxidized analog of alpha-human atrial natriuretic polypeptide is a selective agonist for the atrial-natriuretic-polypeptide clearance receptor which lacks a guanylate cyclase.

The differences in biological functions between alpha-human atrial natriuretic polypeptide (alpha-hANP) and its oxidized analog, MetSO-alpha-hANP, have been investigated. Analysis of the ANP receptor subtypes by affinity labeling has shown that a bovine pulmonary aortic endothelial cell line (CPAE cells) primarily expresses ANP-R1 (R, receptor) coupled to particulate guanylate cyclase, while Hela cells from human cervical carcinoma predominantly express ANP-R2, which lacks a guanylate cyclase. alpha-hANP could bind to both ANP receptor subtypes with high affinity, while MetSO-alpha-hANP showed more selective binding to ANP-R2 than to ANP-R1. The activity of MetSO-alpha-hANP for stimulation of guanylate cyclase coupled to ANP-R1 was about 520-fold less than that of alpha-hANP (median effective dose = 2.5 nM for alpha-hANP, 1.3 microM for MetSO-alpha-hANP), indicating that MetSO-alpha-hANP was a partial agonist for this receptor. While this oxidized analog could inhibit the cAMP production through ANP-R2, with 0.15 times the activity of alpha-hANP (median concentration = 0.31 nM for alpha-hANP, 2.0 nM for MetSO-alpha-hANP). In in vivo studies, the diuretic activity of MetSO-alpha-hANP was 25-100-fold less than that of alpha-hANP. In addition, MetSO-alpha-hANP could potentiate the diuretic activity of alpha-hANP that was also caused by C-ANF4-23, a specific agonist for ANP-R2. These results demonstrate that MetSO-alpha-hANP can act as an agonist more selective for ANP-R2 than for ANP-R1, both in vivo and in vitro. The relationship between receptor selectivities and the conformation of alpha-hANP or MetSO-alpha-hANP was also discussed.

Affinity Labels↗
PubMed1992

Conformational study of endothelins and sarafotoxins with the cystine-stabilized helical motif by means of CD spectra.

A series of CD measurements were carried out on members of peptides in the endothelin and sarafotoxin families. The helical structures taken by these peptides containing the helical motif with the sequences of Cys-X-X-X-Cys and Cys-X-Cys [Y. Kobayashi et al. (1991) Neurochemistry International Vol. 18, pp. 525-534] are classified into three groups: a group of structures of ET-1, ET-2 and vasoactive intestinal contractor (VIC), a group of sarafotoxin, and a group of ET-3.

Amino Acid Sequence↗
PubMed1992

1H-NMR assignment and secondary structure of human insulin-like growth factor-I (IGF-I) in solution.

Human insulin-like growth factor-I (IGF-I) was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for all the backbone protons and almost all of the sidechain protons of the total 70 amino acid residues, using sequence-specific assignment procedures. The secondary structure elements of human IGF-I were identified by investigation of the sequential and medium range NOEs as a preliminary step in determining the three-dimensional structure of this protein by means of distance geometry calculations. The typical NOEs of d alpha beta(i,i + 3) and d alpha N(i,i + 3), as well as the successive strong NOEs of dNN connectivities and slowly exchanging amide protons confirmed the presence of three helical segments corresponding to the sequence regions, Ala8-Cys18, Gly42-Cys48, and Leu54-Cys61, and the existence of a beta-turn in the Gly19-Gly22 region. Our results definitely indicate that the secondary structure of human IGF-I in solution is consistent with that of insulin in the crystalline state.

Amino Acid Sequence↗