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Biomedical subjects

Y Kyogoku

Publications and source records attributed to Y Kyogoku.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1990

Direct observation of the phase behavior of the lipid bilayers of phage PM2 and the intact host cells by 1H-31P cross-polarization NMR.

A method for obtaining the 31P NMR spectrum of a particular supramolecular structure in an intact biological system was developed by applying the 1H-31P cross-polarization technique to a lipid-containing bacteriophage, PM2, and its host bacterium, Alteromonas espejiana. It was shown that 31P NMR spectra of nucleic acids and lipid bilayers can be obtained separately with short and long thermal contact times, respectively. The temperature dependence of the chemical shift anisotropy (delta sigma = sigma parallel - sigma perpendicular) was examined for the separately obtained membrane spectra. Referring to the results of thermal analysis and 31P NMR spectra of bilayers of the extracted phospholipids, the phase transition of the biomembrane was identified for the PM2 phage and the host cell. The dynamic state of the biomembrane of the intact bacterium was directly monitored in detail. The phase behavior of the PM2 lipid bilayer showed good agreement with the earlier report (Akutsu et al., 1980). It turned out that the phase behavior of the intact biomembrane is different from that of the bilayer of the extracted lipids for both PM2 and the host cell. Namely, the terminal temperatures of the phase transition of the host cell and PM2 membranes were lower and higher than those of the extracted phospholipids, respectively.

Bacteriophage lambda↗
PubMed1990

Structural studies of the contractile tail sheath protein of bacteriophage T4. 1. Conformational change of the tail sheath upon contraction as probed by differential chemical modification.

Differential chemical modifications of tyrosine residues of the tail sheath protein, gp18, were performed to elucidate the structural change of the tail sheath upon contraction. Tyrosine residues of monomeric gp18, extended tail sheath, and contracted tail sheath were nitrated by tetranitromethane, and the modified tyrosine residues in each state of the sheath protein were identified by peptide mapping and amino acid sequence analyses of the isolated peptides. Of 31 tyrosine residues in gp18 monomer or in the extended sheath, 12 or 13 residues (Tyr63 and/or -73, -225, -254, -270, -304, -455, -460, -493, -532, -535, -569, and -590) were modified. When photo-CIDNP difference spectra were measured with monomeric gp18, two peaks, which are due to highly exposed tyrosine residues on the molecular surface of gp18, were observed. These two peaks disappeared when the monomeric gp18 was nitrated. With contracted sheath, however, only eight tyrosine residues (Tyr225, -254, -270, -455, -460, -493, -532, and -535) were nitrated on the contracted sheath. Chemical modification of cysteine residues by sulfhydryl group specific reagent ABD-F [(4-aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole] revealed that, among five cysteine residues, Cys377, Cys477, and Cys607 have a sulfhydryl group. Cys402 and Cys406 were modified only under reducing conditions, which strongly suggested the presence of a disulfide bond between these two residues.

Chemical Phenomena↗
PubMed1990

Estimation of microscopic redox potentials of a tetraheme protein, cytochrome c3 of Desulfovibrio vulgaris, Miyazaki F, and partial assignments of heme groups.

The microscopic formal redox potentials of a tetraheme protein, cytochrome c3 from Desulfovibrio vulgaris, Miyazaki F, were estimated from the chemical shifts of the heme methyl signals in its 1H NMR spectrum. All chemical shifts in the five macroscopic oxidation states were determined for eight of the heme methyl protons by the saturation-transfer method. The electron-distribution probability at each heme in each oxidation state was estimated directly from the chemical shifts. To minimize errors due to interheme pseudocontact contributions, the average electron-distribution probability was used for calculation of the microscopic formal redox potentials. By introducing interacting potentials, 32 parameters were reduced to 10. The 10 parameters were determined analytically from the 9 independent electron-distribution probabilities and 2 macroscopic formal redox potentials. The results showed the presence of a strong positive interaction between a pair of particular hemes. The microscopic formal redox potential changes dramatically with the extent of reduction because of the intramolecular interheme interactions. NMR signals of two hemes were assigned to particular hemes in the crystal structures by nuclear Overhauser effect experiments. The results showed that the hemes with the highest and lowest redox potentials in the one-electron reduction process correspond to hemes I and IV in the crystal structure.

Cytochrome c Group↗
PubMed1990

A new model for the bending of DNAs containing the oligo(dA) tracts based on NMR observations.

The conformations of double-stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) and d(GCATTTTGAAACG)d(CGTTTCAAAATGC) have been studied by NMR spectroscopy. Analyses of cross peaks in NOESY spectra between the H2 of an adenine and the H1' of a deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' in each duplex. In view of this gradual compression of the minor groove along the oligo(dA) tract, it can be understood clearly why d(GGAAATTTCC)n x 2 and d(GAAAATTTTC)n x 2 are bent, and d(GGTTTAAACC)n x 2 and d(GTTTTAAAAC)n x 2 are not bent. The relative extents of bending of a series of d(AjN10-j)nd(N10-jTj)n sequences can also be understood systematically. Additionally, it was found that the TA step disturbed the compression of the minor groove of the oligo(dA) tract to some extent.

Base Sequence↗