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Biomedical subjects

Y Kyogoku

Publications and source records attributed to Y Kyogoku.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Characterization of the bacterially expressed Drosophila engrailed homeodomain.

To investigate the mechanism of DNA recognition by the homeodomain, truncated proteins containing the entire homeodomain encoded by the Drosophila engrailed gene were expressed in Escherichia coli. Each protein was accumulated to an amount representing more than 40% of the total bacterial protein and recovered in the soluble fraction. Of the three truncated proteins, the shortest one (71 amino acid residues) was further purified by conventional chromatography. The purified engrailed homeodomain (En-HD) protected a DNA sequence, TTAATT, the core element of consensus sequences recognized by many other homeodomain proteins, from DNase I digestion. UV-CD spectra of the En-HD showed that it mainly consisted of alpha-helix. Based on one-dimensional 1H-NMR spectra, the tertiary structure of the En-HD was shown to be stable against temperature up to 50 degrees C and low pH. The low pH resistance of the protein was also demonstrated by UV-CD measurement. Thus, the current over-production system provides an active and stable homeodomain, which is suitable for structure-function analysis.

Animals↗
PubMed1992

Fission yeast pap1-dependent transcription is negatively regulated by an essential nuclear protein, crm1.

The fission yeast pap1+ gene encodes an AP-1-like transcription factor that contains a leucine zipper motif. We identified a target gene of pap1, the p25 gene. The 5' upstream region of the p25 gene contains an AP-1 site, and by DNase I footprint analysis, we showed that the pap1 protein binds to the AP-1 site as well as to a 14-bp palindrome sequence. p25 is overproduced when the pap1+ gene is overexpressed, whereas p25 is not produced at all in the pap1 deletion mutant. p25 was previously found to be overproduced in strains carrying cold-sensitive crm1 mutations whose gene product is essential for viability and is thought to play an important role in maintenance of a proper chromosomal architecture. Deletion and site-directed mutagenesis of sequences upstream of the p25 gene demonstrated that the AP-1 site as well as the palindrome sequence are crucial for transcriptional activation either by pap1 overproduction or by the cold-sensitive crm1 mutation; pap1+ is apparently negatively regulated by crm1+. Moreover, we found that cold-sensitive crm1 mutations are suppressed by the deletion of pap1+, further indicating a close relationship between crm1+ and pap1+. The crm1 protein is highly conserved; the budding yeast homolog, CRM1, which complements the fission yeast cold-sensitive crm1 mutation, was isolated and found to also be essential for viability. These results suggest the functional importance of chromosome structure on the regulation of gene expression through the pap1 transcription factor.

Amino Acid Sequence↗
PubMed1991

Identification of transcriptional suppressor proteins that bind to the negative regulatory element of the human immunodeficiency virus type 1.

Two different proteins which independently bound to neighboring sequences within the negative regulatory element (NRE) of human immunodeficiency virus type 1 (HIV-1) were detected in the nuclear extract of a virus-infected human T cell line. One of the factors bound to a novel dyad symmetrical sequence. This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene. Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay. The other factor bound to a putative AP-1 recognition sequence predicted for the HIV-1 NRE. However, this factor did not bind to a typical AP-1 site. The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third, respectively. Thus, each factor may function as a novel negative regulator of transcription.

Base Sequence↗
PubMed1991

Nuclear magnetic resonance study on the structure and interaction of cyclic AMP receptor protein and its mutants: a deuterium-labeling and photo-CIDNP study.

The identification and assignment of the proton magnetic resonances of some aliphatic and aromatic amino acid residues of cyclic AMP receptor protein (CRP) are reported. The signals of the leucine and valine residues at around 0 ppm were identified on the basis of intermolecular nuclear Overhauser effects, deuterium labeling, and partial proteolytic digestion. On the addition of cAMP, methyl proton signals due to Val-49 and three leucine residues were detected as upfield-shifted signals at around -0.2 ppm. These signals can be used as indicators of the proper binding of cAMP because they are not observed on the addition of cGMP or 2'-deoxy-cAMP. They are also not observed on cAMP binding to mutant CRP*5 (Ser-62-Phe), which can only be activated by a high concentration of cAMP, but they are observed on cAMP binding to other mutant CRP*s (four species), which can be activated by lower concentrations of cAMP. The resonance of some aromatic protons, i.e., C-2H of two tryptophans, C-2H and C-4H of six histidines, and C-2,6H and C-3,5H of six tyrosine residues in CRP, were assigned by means of deuterium labeling and NOE measurements. The 1H NMR spectrum of labeled CRP [Trp(ring-d5), Phe(ring-d5), and Tyr(3,5-d2)] showed good resolution in the aromatic region. The addition of cAMP to this CRP in D2O caused pronounced line broadening of resonances arising from the residues in the cAMP-binding domain, but the resonances of the DNA-binding domain were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Spectroscopic studies on lambda cro protein-DNA interactions.

Spectroscopic (circular dichroism and fluorescence) and thermodynamic studies were conducted on lambda Cro-DNA interactions. Some base substitutions were introduced to the operator and the effects on the conformation of the complex and thermodynamic parameters for dissociation of the complex were examined. It was found that, (1) in the specific binding of Cro with DNA which has a (pseudo) consensus sequence, DNA is overwound, while in non-specific binding it is unchanged, or rather unwound; (2) substitution of central base-pairs or the introduction of a mismatched base-pair at the center of the operator reduces the extent of DNA conformational change on Cro binding and lessens the stability of the Cro-DNA complex, even though there is apparently no direct interaction between Cro and DNA at these positions; (3) stability of the complex increases with the degree of DNA conformational change of the same type during binding; (4) in some cases of specific binding, there are three states in the dissociation of the complex as observed by salt titration: two conformational states for the complex depending on salt concentration and, in non-specific binding, dissociation is a two-state transition; (5) the number of ions involved in interactions between Cro and 17 base-pair DNA is about 7.7 for NaCl titrations; (6) dissociation free energy prediction of the Cro-DNA complex by simple addition of the dissociation free energy change of a single base-pair substitution agrees with our experimental results when DNA overwinding occurs during binding, i.e. in specific binding.

Base Sequence↗
PubMed1991

The cystine-stabilized alpha-helix: a common structural motif of ion-channel blocking neurotoxic peptides.

Neurotoxic peptides from venoms of scorpions and honey bees exhibit a consensus pattern in the two disulfide bridgings related to the sequence portions Cys-X-Cys and Cys-X-X-X-Cys. A revised three-dimensional structure of charybdotoxin, as determined by two-dimensional nmr spectroscopy, confirms that the consensus cystine dislocation generates in all these toxins a common structural element, i.e., the cystine-stabilized alpha-helical (CSH) motif, which may be correlated with their common ion channel blocking activity.

Amino Acid Sequence↗
PubMed1991

Assignments of 1H-15N magnetic resonances and identification of secondary structure elements of the lambda-cro repressor.

The assignments of 1H-15N magnetic resonances of the lambda-cro repressor are presented. Individual 15N-amino acids were incorporated into the protein, or it was uniformly labeled with 15N. For the 13C-15N double-labeling experiments, 13C-amino acids were incorporated into the uniformly 15N-labeled protein. All the amide 1H-15N resonances could be assigned with such specific labeling, and sequential connectivities obtained by two-dimensional (2D) 1H-15N reverse correlation spectroscopies and three-dimensional (3D) 1H/15N NOESY-HMQC spectroscopy. Conventional 2D 1H-1H correlation spectroscopies were applied to the assignment of the side-chain protons. Some of the 1H resonance assignments are inconsistent with those previously reported [Weber, P.L., Wemmer, D.E. and Reid, B.R. (1985) Biochemistry, 24, 4553-4562]. The sequential NOE connectivities and H-D exchange rates indicate several elements of the secondary structure, including alpha-helices consisting of residues 8-15, 19-25 and 28-37, and three extended strands consisting of residues 4-7, 39-45 and 49-55. Based on several long-range NOEs, the three extended strands could be combined to form an antiparallel beta-sheet. The amide proton resonances of the C-terminal residues except Ala66 (residues 60-65) were hardly observed at neutral pH, indicating that the arm is flexible. The identified secondary structure elements in solution show good agreement with those in the crystal structure of the cro protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y. and Matthews, B.W. (1981) Nature, 290, 754-758].

Amino Acid Sequence↗
PubMed1991

Preliminary nuclear magnetic resonance studies on human saliva.

Of gustatory-stimulated human whole, parotid, submandibular and sublingual saliva only parotid saliva, a serous rather than mucous secretion, presented a relatively well-resolved proton NMR spectrum with satisfactory signal-to-noise ratio in a short time (30 min). The proton signal intensities showed significant circadian rhythms related to the circadian rhythms of protein concentrations in saliva. Age- and sex-associated differences in spectra were not observed for healthy saliva. On the other hand, marked differences in the spectra were observed for patients with suspected sialoadenitis.

Adult↗
PubMed1991

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations.

The structure of endothelin-1 (ET-1), an endothelial cell-derived peptide with vasoconstricting activity, was determined in an aqueous solution by means of a combination of NMR and distance geometry calculations. The resulting structure is characterized by an alpha-helical conformation in the sequence region, Lys9-Cys15. Furthermore, an extended structure and a turn structure exist in the Cys1-Ser4 and Ser5-Asp8 regions respectively, and no preferred conformation was found for the C-terminal part of the peptide which was not uniquely constrained by the NMR data. These structural elements, the alpha-helical structure in the sequence portion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys, are homologous to those found commonly in several neurotoxic peptides.

Algorithms↗
PubMed1991

Intersubunit disulfide-bonded lambda-Cro protein.

Site-directed mutagenesis has been employed to substitute cysteine for valine at position 55, which is located on the dimer interface of the Cro protein of bacteriophage lambda. It has been found that the Cys55 Cro protein (Cro VC55) spontaneously forms a stable disulfide-bonded dimer in the absence of a reducing agent. UV-CD and NMR data showed that the mutant protein retains the conformation of the wild Cro protein and has acquired significant heat-stability. However, its specific DNA-binding activity is reduced several times compared with that of the wild Cro. Photochemically induced dynamic nuclear polarization (CIDNP) spectra demonstrated that a conformational change of Cro VC55 did not take place upon the formation of a complex with OR3, in contrast to the case of the wild Cro. These data suggest that the induced fitting, like loosening, of the two subunits of the wild Cro dimer contributes to the enhancement of its affinity to its operator DNA, which results in a specific interaction between Cro and OR3.

Base Sequence↗
PubMed1991

Structure analyses of DNA oligomers by use of heteronuclear 2D NMR.

DNA oligomer d(CGGAAGACTCTCCTCCG):d(CGGAGGAGAGTCTTCCG) named UASG (17mer M.W. = 11 kDa) was studied by 1H NMR and heteronuclear two dimensional (2D) NMR. All the labile protons and half of the non-exchangeable protons were assigned by use of conventional 1H 2D experiments including NOESY using 1-1 echo excitation for water suppression. Signal degeneracy in the sugar proton region made it difficult to make assignments of the remaining half of the non-exchangeable protons of the oligomer in 1H 2D spectra. Here we report a new strategy using 1H/13C and 1H/31P heteronuclear single-quantum correlation spectroscopy combined with homonuclear three dimensional NOESY-TOCSY. By this strategy, most of the proton resonances of the oligomer have been assigned, and it turned out that the whole conformation of the oligomer is B-form like.

Base Sequence↗
PubMed1991

An NMR study on the conformation of a deoxyoligonucleotide duplex, d(GGGGCCCC)2, and its complex with chromomycin.

The conformation of drug-free d(GGGGCCCC)2 and the chromomycin-d(GGGGCCCC)2 complex in aqueous solution were studied by NMR spectroscopy. The present study has indicated that free d(GGGGCCCC)2 takes the B form in solution, although it takes the A form in the crystalline state. The NMR spectrum of the complex indicated that chromomycin binds as a symmetry-related dimer to the minor groove of the central four residues of d(GGGGCCCC)2. The drastic conformational change in the central four residues of d(GGGGCCCC)2 on going from the B form family to the A form was demonstrated by the characteristic NOEs and coupling patterns. The change seems to be indispensable for accommodation of the bulky chromomycin dimer in the minor groove. On the basis of the intermolecular NOEs between chromomycin and d(GGGGCCCC)2, the structure of the complex has been constructed and refined by energy minimization.

Base Sequence↗
PubMed1990

Determination of the conformation of d(GGAAATTTCC)2 in solution by use of 1H NMR and restrained molecular dynamics.

The conformation of the putative bent DNA d(GGAAATTTCC)2 in solution was studied by use of 1H NMR and restrained molecular dynamics. Most of the resonances were assigned sequentially. A total of 182 interproton distance restraints were determined from two-dimensional nuclear Overhauser effect spectra with short mixing times. Torsion angle restraints for each sugar moiety were determined by qualitative analysis of a two-dimensional correlated spectrum. Restrained molecular dynamics was carried out with the interproton distances and torsion angles incorporated into the total energy function of the system in the form of effective potential terms. As initial conformations for restrained molecular dynamics, classical A-DNA and B-DNA were adopted. The root mean square deviation (rmsd) between these two conformations is 5.5 A. The conformations obtained by use of restrained molecular dynamics are very similar to each other, the rmsd being 0.8 A. On the other hand, the conformations obtained by use of molecular dynamics without experimental restraints or restrained energy minimization depended heavily on the initial conformations, and convergence to a similar conformation was not attained. The conformation obtained by use of restrained molecular dynamics exhibits a few remarkable features. The second G residue takes on the BII conformation [Fratini, A. V., Kopka, M. L., Drew, H. R., & Dickerson, R. E. (1982) J. Biol. Chem. 257, 14686-14707] rather than the standard BI conformation. There is discontinuity of the sugar puckering between the eighth T and ninth C. The minor groove of the oligo(dA) tract is rather compressed. As a result, d(GGAAATTTCC)2 is bent.

Magnetic Resonance Spectroscopy↗