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Biomedical subjects

Y Kyogoku

Publications and source records attributed to Y Kyogoku.

At least 109 records · Page 6Linked to original sources
PubMed1990

Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae.

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred in the presence of 3'AMP. 1H-NMR titration and photo-chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and His115 were partially modified to yield a total of one mole of N tau-carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or His115 inactivates the enzyme.

Amino Acid Sequence↗
PubMed1990

A nuclear magnetic resonance study of the cyclic AMP receptor protein (CRP): assignments of the NH protons of histidine and tryptophan residues and the effect of binding of cAMP to CRP.

Seven well-resolved signals could be observed in the field lower than 9.5 ppm in the 1H-NMR spectrum of the H2O solution of cyclic AMP receptor protein (CRP). The signals of the tryptophan and histidine residues were identified on the basis of the CPMG spin echo spectra, the intra-residue NOE, 15N labeling, deuterium labeling, and the results of pH titration. The assignments of peaks to specific tryptophan and histidine residues are discussed in relation to the amino acid sequence and X-ray crystallographic data, and were confirmed by experiments involving partial subtilisin digestion. The four signals E (11.25 ppm), F (11.15 ppm), G (10.75 ppm), and H (10.65 ppm) were tentatively assigned to the resonances of the histidine residue at position 159, the arginine residue at position 82, and the tryptophan residues at positions 85 and 13, respectively. On the addition of cAMP and cGMP, signals F and G shifted up- and downfield respectively and conformational changes in the structure of CRP could be detected. The conformational transition mostly occurs when one cAMP molecule binds to one of the dimer subunits, but is completed only when both cAMP binding sites are saturated.

Carrier Proteins↗
PubMed1990

Physiological dynamic structures of nucleic acids in A. espejiana cells detected on 1H-31P cross-polarization NMR.

In order to obtain information of the supramolecular structures of nucleic acids in vivo, the 1H-31P cross-polarization NMR technique was applied to intact marine bacterial cells. An asymmetric powder pattern spectrum of nucleic acids in the cell was observed. The major contributor to the spectrum was ribosomes. Furthermore, the powder pattern changed dramatically with the physiological conditions of the cells. The results showed that this is a promising method for the investigation of the supramolecular structures of nucleic acids in vivo.

Bacteria, Aerobic↗
PubMed1990

Efficient expression and Zn(II)-dependent structure of the DNA binding domain of the yeast GAL4 protein.

Three protein fragments of different sizes which contain the DNA binding domain of transcription factor GAL4 from Saccharomyces cerevisiae have been expressed in functional forms in Escherichia coli. DNase I footprinting and gel retardation assays showed that the purified proteins bound to the same DNA sequence on the gal1-gal10 promoter as intact GAL4 does. Denaturation--refolding experiments demonstrated that Zn(II) is necessary for maintenance of the conformation of the DNA binding domain of GAL4, as judged on UV-CD and 1H-NMR measurements, as well as for specific DNA binding.

Amino Acid Sequence↗
PubMed1990

The differences in conformation between alpha-human atrial natriuretic polypeptide, alpha-hANP, and its derivative, Met(O)-alpha-hANP, in solution.

The differences in conformation between alpha-human atrial natriuretic polypeptide (alpha-hANP) and its inactive analog, Met(O)-alpha-hANP, have been analyzed by nuclear magnetic resonance spectroscopy. All proton resonances for both peptides were assigned by means of the sequential assignment procedure. The three-dimensional structure of alpha-hANP in solution had previously been determined by distance geometry calculation using distance constraints derived from nuclear Overhauser effects (NOEs). Here, the three-dimensional structure of Met(O)-alpha-hANP was determined. The conformational differences between these two molecules were as follows: three segments of alpha-hANP, Ser1-Cys7, Arg11-Ala17 and Gln18-Tyr28, have some ordered structures. In Met(O)-alpha-hANP the Gln18-Tyr28 region has a similar conformation, while the remaining two regions do not have the ordered structure found in alpha-hANP. It is suggested that the conserved conformation of the Gln18-Tyr28 region is required for binding to the ANP receptor and that the slight biological activity of Met(O)-alpha-hANP is due to loss of the ordered structures evoked in the Ser1-Cys7 and Arg11-Ala17 regions of alpha-hANP.

Amino Acid Sequence↗
PubMed1990

Assignments of the iminoproton resonances of Bombyx mori tRNA(UCCGly) and the comparison of its structure and stability with those of tRNA(GCCGly).

Most of the iminoproton resonances in the 1H-NMR spectrum of Bombyx mori tRNA(UCCGly) have been assigned by the sequential NOEs. Any peak which indicates the presence of the tertiary GC base pair between the D and T loops could not be detected. The effects of temperature and the addition of magnesium ions and spermine on the 1H-NMR spectrum of this tRNA were examined. From the temperature change, it was found that the acceptor stem and the D stem in Bombyx mori tRNA(UCCGly) are equally stable even in the absence of magnesium, which is different from tRNA(GCCGly) where the D stem is not so stable.

Animals↗
PubMed1990

An NMR study on the conformation of the chromomycin-d(GGGGCCCC)2 complex.

The conformation of the chromomycin-d(GGGGCCCC)2 complex in aqueous solution was studied by NMR spectroscopy. The NMR spectrum of the complex indicated that the 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 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.

Chromomycins↗
PubMed1989

Solution conformation of conotoxin GI determined by 1H nuclear magnetic resonance spectroscopy and distance geometry calculations.

Conformational analysis of conotoxin GI, one of the neurotoxic peptides produced by a marine snail, genus Conus, was performed by a combination of nuclear magnetic resonance spectroscopy (NMR) and distance geometry calculations. The resulting conformers on minimization of the target function were classified into two groups. The difference in the structures of the conformers is mainly due to the difference in the orientation of the side chain of the tyrosyl residue. The results show that the solution structure of conotoxin GI satisfies the conformational requirements for the biological activity of an antagonist toward nicotinic cholinergic receptors elucidated in a series of studies on alkaloids. The structure is discussed on the basis of the results of comparison of the atomic arrangements of the active sites of snake venom peptides and molecular models based on the results of secondary structure prediction.

Amino Acid Sequence↗
PubMed1989

Photo-CIDNP studies of Bence Jones proteins, immunoglobulins, and their proteolytic fragments.

The results of photo-CIDNP measurements of Bence Jones proteins, immunoglobulins, and their proteolytic fragments are reported. The CIDNP spectra of constant region CL fragments, which were derived from lambda-type Bence Jones proteins with three different isotypes, gave polarizations originating from two Tyr and one Trp residues. From comparisons of the results obtained by using lambda-type Bence Jones proteins that possess the known amino acid sequences with those for the CL fragments, it was concluded that the CIDNP signals observed in the case of Bence Jones proteins are all due to the hypervariable region. On the basis of the results obtained by using IgG1, IgG2, IgG3, and IgG4 myeloma proteins along with their Fab, Fc, and pFc' fragments, it was concluded that CIDNP signals for the Fab region all originate from the variable region. The Fc region gives two Tyr polarizations, one of which has been assigned to Tyr-296. We have also shown that the second Tyr signal is due to Tyr-373. Interaction of Fc and staphylococcal protein A has also been examined by the CIDNP technique.

Amino Acid Sequence↗
PubMed1989

A new model for DNA bending on the basis of gradual compression of the minor groove from 5' to 3' along the oligo(dA) tract.

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 means of NMR spectroscopy. Analyses of cross peaks in NOESY spectra between H2 of an adenine and H1' of the 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' along the tract in four oligonucleotides. A new model is proposed as to DNA bending based on the evidence of gradual compression of the minor groove. This model can explain 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 bending of d(AjN10-j) n x 2 sequences can also be explained.

DNA↗
PubMed1989

Creation of a nuclear magnetic resonance data repository and literature database.

We believe the need exists for an organized and accessible repository of protein nuclear magnetic resonance data. The structure and dynamics of hundreds of proteins are being investigated by NMR. NMR data currently are spread throughout the world and often are not published for lack of journal space. Difficulties in locating, obtaining, and correlating these data with protein structures limits their usefulness to the scientific community. In time, the data may become lost or ignored. To provide a collection point for the results of protein NMR studies and a uniform means of distributing these data, we propose that a data bank be created consisting of two databases: a comprehensive and thoroughly indexed database for the NMR literature and a data repository for the storage of extensive protein NMR data sets. Our current specifications for the types of information to be stored in the NMR databases and their organization for dissemination are defined. The design is intended to be flexible, capable of expanding to include new techniques, new forms of data, and biopolymers other than proteins. This is a proposal to the community of NMR spectroscopists. Only through the active cooperation and support of those in the field of NMR spectroscopy can the proposed data bank succeed.

Database Management Systems↗
PubMed1988

A 2H-NMR study on the glycerol backbone of phospholipids extracted from Escherichia coli grown under high osmotic pressure: evidence for multiconformations of phosphatidylethanolamine.

A glycerol-requiring auxotroph was isolated from mutagenized Escherichia coli K-12 UFAts cells. This auxotroph was used for the specific deuteration of E. coli phospholipids. The cells were grown under high osmotic pressure (in the presence of 2.0% KCl). The membrane had a highly saturated fatty acid composition (76% phosphatidylethanolamine, 20% cardiolipin and 4% phosphatidylglycerol). The deuterium magnetic resonance spectra of coarse liposomes of the extracted phospholipids with perdeuterated glycerol incorporated into them were measured. To obtain well characterized information, phospholipid mixtures reconstituted from the deuterated and nondeuterated components at the same ratios as in the case of the total extract were used. On the analysis of the spectra, the following conclusions were drawn. (1) The whole polar region of cardiolipin is dynamically symmetric and quite rigid in the presence of phosphatidylethanolamine. (2) Although the quadrupole splittings of the deuterons at the C-2 and C-3 positions of the glycerol backbone were similar to each other, those at the C-1 position for phosphatidylethanolamine and cardiolipin are different, even in the same bilayer. (3) Furthermore, each C-1 deuteron of phosphatidylethanolamine gave rise to a doublet, suggesting the presence of two backbone conformations, between which there is slow exchange. (4) The polar head group of phosphatidylethanolamine interacts with cardiolipin and phosphatidylglycerol in different ways, which could be responsible for the different osmotic properties of the vesicles composed of them.

Cardiolipins↗
PubMed1988

One- and two-dimensional NMR studies on the conformation of DNA containing the oligo(dA)oligo(dT) tract.

The resonances of the imino protons and all of the non-exchangeable protons (except for H5'/H5'') of d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) have been assigned by means of one- and two-dimensional NMR spectroscopies. Qualitative analyses showed that the overall structure is of the B-form, but local conformational deviations exist. The NOEs between the imino protons of thymines and H2 of adenines suggest that the A-T base pairs are propeller-twisted to almost the same degree as in crystals. A remarkable chemical shift of H1' was observed for the residue located just before the oligo(dA)oligo(dT) tract, suggesting the presence of conformational discontinuity at the junctions between the oligo(dA)oligo(dT) tract and the other portions. Analyses of cross peaks in NOESY spectra between H2 of adenines and H1' of the 3'-neighbouring residues on the complementary strand revealed that the minor groove of the oligo(dA)oligo(dT) tract is narrow and compressed gradually, from 5' to 3', along the tract.

Base Sequence↗
PubMed1988

A photo-chemically induced dynamic nuclear polarization NMR study on rabbit and bovine cytochrome b5.

Although it has been indicated that proteins with chromophoric groups are not suitable for photo-chemically induced dynamic nuclear polarization (photo-CIDNP) measurements, we have successfully obtained these spectra for a heme protein, cytochrome b5. The characteristics of the spectra differed in some points from those so far reported. The intensities of the signals in the aromatic region were very weak, while those of the beta-methylene protons of one histidine and one tryptophan were extremely strong in comparison with the aromatic protons. It was demonstrated, on the basis of the photo-CIDNP spectrum, that one of seven histidines, all three tyrosines and a single tryptophan of the rabbit soluble cytochrome b5 are exposed on the surface of the protein. The results of comparison of the photo-CIDNP spectra for the rabbit soluble and intact, and bovine intact, cytochrome b5 led us to the conclusion that the conformation of the hydrophilic, catalytic part of cytochrome b5 is quite similar among these three proteins. In the presence of Chaps micelles, bovine intact cytochrome b5 was in monomeric form and the histidine signals disappeared from its photo-CIDNP spectrum. When bovine intact cytochrome b5 was reconstituted into egg yolk phosphatidylcholine liposomes, although separate signals due to the protein part were observed in the normal 1H-NMR spectrum, no photo-CIDNP signal could be detected. The normal spectrum suggests that the conformation of the protein embedded in liposomes is similar to that of the oligomeric form without lipids or a detergent.

Animals↗
PubMed1988

Hydrogen-exchange kinetics of the indole NH proton of the buried tryptophan in the constant fragment of the immunoglobulin light chain.

The constant fragment of the immunoglobulin light chain (type lambda) has two tryptophyl residues at positions 150 and 187. Trp-150 is buried in the interior, and Trp-187 lies on the surface of the molecule. The hydrogen-deuterium exchange kinetics of the indole NH proton of Trp-150 were studied at various pH values at 25 degrees C by 1H nuclear magnetic resonance. Exchange rates were approximately first order in hydroxyl ion dependence above pH 8, were relatively independent of pH between pH 7 and 8, and decreased below pH 7. On the assumption that the exchange above pH 8 proceeds through local fluctuations of the protein molecule, the exchange rates between pH 7 and 8 through global unfolding were estimated. The exchange rate constant within this pH range at 25 degrees C thus estimated was consistent with that of the global unfolding of the constant fragment under the same conditions as those reported previously [Kikuchi, H., Goto, Y., & Hamaguchi, K. (1986) Biochemistry 25, 2009-2013]. The activation energy for the exchange process at pH 7.8 was the same as that for the unfolding process by 2 M guanidine hydrochloride. The exchange rates of backbone NH protons were almost the same as that of the indole NH proton of Trp-150 at pH 7.1. These observations also indicated that the exchange between pH 7 and 8 occurs through global unfolding of the protein molecule and is rate-limited by the unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Bence Jones Protein↗
PubMed1988

Structures of sugar chains of ricin D.

The toxic lectin, ricin D, contains mannose, fucose, xylose, and N-acetylglucosamine as sugar components. Sugar chains are linked to Asn-10 of the A-chain, and to Asn-95 and Asn-135 of the B-chain (Funatsu, G. et al. (1978) Agric. Biol. Chem. 42, 501-503; Araki, T. & Funatsu, G. (1985) FEBS Lett. 191, 121-124). Asparagine-linked sugar chains of each glycopeptide from ricin D were liberated by hydrazinolysis followed by N-acetylation. The reducing end residues of the sugar chains were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives obtained were purified by gel-filtration and reversed-phase HPLC. Eight main PA-sugar chains were obtained from three glycopeptides and the structures of these sugar chains were determined by component analysis, stepwise exoglycosidase digestions, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. The results show that oligomannose type sugar chains (Man6-7GlcNAc2) are linked to Asn-95; Man5-7 GlcNAc2 and M4X (structure, see below) to Asn-135 of the B-chain, and M3FX and M3X to Asn-10 of the A-chain. (Formula: see text).

Acetylation↗
PubMed1988

The conformation of alpha-human atrial natriuretic polypeptide in solution.

The three-dimensional structure of alpha-human ANP in solution was determined through the combined use of nuclear magnetic resonance spectroscopy and distance geometry. The results are based on distance constraints determined by nuclear Overhauser effect measurements and one disulfide bond. The structure is as follows. Three separate regions, which are Ser1-Cys7, Arg11-Ile15, and Gln18-Tyr28 each have some ordered structure. The remaining parts in the sequences of Gly9-Gly10 and Gly16-Ala17 act as hinges. And the C-terminal part is folded back toward the cyclic moiety. The conformation of alpha-hANP reported here is expected to give a better understanding of the relationships between its biological activities and three-dimensional structure.

Amino Acid Sequence↗