268 results on '"Masatsune Kainosho"'
Search Results
52. Slow Motion in the CAA·TTG Sequence of a DNA Decamer Duplex Studied by NMR
- Author
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Thomas L. James, Masatsune Kainosho, Nikolai B. Ulyanov, and Chojiro Kojima
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Stereochemistry ,Guanosine ,Crystal structure ,Biochemistry ,Oligomer ,chemistry.chemical_compound ,Moiety ,A-DNA ,Base Pairing ,Nuclear Magnetic Resonance, Biomolecular ,Purine Nucleotides ,Carbon Isotopes ,Base Sequence ,Nitrogen Isotopes ,Chemistry ,Relaxation (NMR) ,Nucleic Acid Heteroduplexes ,DNA ,Crystallography ,Oligodeoxyribonucleotides ,Duplex (building) ,Nucleic Acid Conformation ,Thermodynamics ,Pyrimidine Nucleotides ,Imines ,Protons - Abstract
In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA*TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by 1H, 13C, and 15N NMR relaxation measurements on a DNA decamer d(CATTTGCATC)*d(GATGCAAATG). The DNA decamer was enriched with 15% 13C and 98% 15N isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA*TTG moiety. Analysis of (15)N relaxation showed that the order parameter, S2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer. The strong temperature dependence of guanosine NH S2 in the CAA*TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA*TTG sequence were broadened in 2D 1H NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the 1H T2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG*CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA*TTG sequence but extending over the CAAAT*ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the 1H, 13C, and 15N T1rho values. 1H and 13C T1rho values showed a pulse field strength dependence, but 15N T1rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 micros was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.
- Published
- 2001
53. [Untitled]
- Author
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Masatsune Kainosho, Akira Ono, Ad Bax, and Zhengrong Wu
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Coupling ,Bond length ,Crystallography ,Dodecameric protein ,Hydrogen ,Chemistry ,Residual dipolar coupling ,Hydrogen bond ,chemistry.chemical_element ,Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid ,Biochemistry ,Spectroscopy ,Magnetic dipole–dipole interaction - Abstract
The ratio of the internucleotide dipolar coupling and the corresponding one-bond imino 15 N- 1 H dipolar coupling provides a measure for the N H/H-N distance ratio. Measurements were carried out for a dodecamer, d(CGCGAATTCGCG)2, in which a C-G and an A-T basepair were uniformly enriched in 15 N. When assuming H-bonds to be perfectly linear, dipolar data indicate time-averaged hydrogen bond lengths of 1:80 0:03 A for A-T and 1:86 0:02 A for C-G. When using H-bond orientations from high resolution X-ray data, H-bond lengths are about 0.1 A shorter.
- Published
- 2001
54. [Untitled]
- Author
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Chojiro Kojima, Etsuko Kawashima, Yoshiharu Ishido, Akira Ono, Takeshi Sekine, Yoshimasa Kyogoku, and Masatsune Kainosho
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Coupling constant ,chemistry.chemical_classification ,Karplus equation ,Deuterium ,Proton ,chemistry ,Stereochemistry ,A-DNA ,Nucleotide ,Dihedral angle ,J-coupling ,Biochemistry ,Spectroscopy - Abstract
The sugar conformation of a DNA decamer was studied with proton-proton 3J coupling constants. Two samples, one comprising stereospecifically labeled 2′-R-2H for all residues and the other 2′-S-2H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific ≥ 99% for all 2′′-2H, ≥ 98% for 2′-2H of A, C, and T, and ≥ 93% for 2′-2H of G). The 3J values of all H1′-H2′ and H1′-H2′′ pairs, and several H2′-H3′ and H2′′-H3′ pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3′-exo and C2′-endo with Φm values of 26° to 44°, except for the second and 3′ terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from JH1′H2′ and JH1′H2′′ values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1′-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.
- Published
- 2001
55. [13C,13C]- and [13C,1H]-TROSY in a Triple Resonance Experiment for Ribose−Base and Intrabase Correlations in Nucleic Acids1
- Author
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Konstantin Pervushin, César Fernández, Roland Riek, Masatsune Kainosho, and Kurt Wüthrich
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chemistry.chemical_classification ,Spins ,Resonance ,General Chemistry ,Ring (chemistry) ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Nuclear magnetic resonance ,chemistry ,Ribose ,Nucleic acid ,Nucleotide ,Anisotropy ,Spectroscopy - Abstract
A novel TROSY (transverse relaxation−optimized spectroscopy) element is introduced that exploits cross-correlation effects between 13C−13C dipole−dipole (DD) coupling and 13C chemical shift anisotropy (CSA) of aromatic ring carbons. Although these 13C−13C effects are smaller than the previously described [13C,1H]-TROSY effects for aromatic 13C−1H moieties, their constructive use resulted in further transverse relaxation−optimization by up to 15% for the resonances in a 17 kDa protein−DNA complex. As a practical application, two- and three-dimensional versions of the HCN triple resonance experiment for obtaining ribose−base and intrabase correlations in the nucleotides of DNA and RNA (Sklenar, V.; Peterson, R. D.; Rejante, M. R.; Feigon, J. J. Biomol. NMR 1993, 3, 721−727) have been implemented with [13C,1H]- and [13C,13C]-TROSY elements to reduce the rate of transverse relaxation during the polarization transfers between ribose 13C1‘ and base 15N1/9 spins, and between 13C6/8 and N1/9 within the bases. The...
- Published
- 2001
56. Developing model systems for the NMR study of substituent effects on the N?H���N hydrogen bond in duplex DNA
- Author
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Rei Ishikawa, Chojiro Kojima, Masatsune Kainosho, and Akira Ono
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Proton ,Base pair ,Hydrogen bond ,Substituent ,General Chemistry ,Crystallography ,chemistry.chemical_compound ,Dodecameric protein ,chemistry ,Proton NMR ,Organic chemistry ,General Materials Science ,Thymidine ,DNA - Abstract
The substitution effects on various parameters, which may influence the hydrogen bond strengths of Watson–Crick-type base pairs, were investigated for DNA dodecamers containing 5-substituted-2′-deoxyuridine derivatives. In doing so, a series of [3-15N]-2′-deoxyuridine derivatives, including thymidine, 2′-deoxyuridine, 5-bromo-2′-deoxyuridine, 5-fluoro-2′-deoxyuridine and 5-cyano-2′-deoxyuridine, and [ul-15N]-2′-deoxyadenosine, were synthesized and incorporated into the DNA dodecamer, d(CGCGA* ATX* CGCG)2, where X* and A* were a [3-15N]-2′-deoxyuridine derivative and [ul-15N]-2′-deoxyadenosine, respectively. The imino proton chemical shift and the spin coupling constant between the imino proton and nitrogen [1J(N,H)] were measured for the Watson–Crick-type A*–X* base pair of all five duplexes. The substitution with an electron-withdrawing group results in downfield shift of the imino proton and a concomitant decrease in magnitude of the 1J(N,H) value, and a good, linear correlation was found between the two effects. These substitution effects on the NMR parameters were linearly correlated with the pKa values of the 2′-deoxyuridine derivatives and also with the theoretically calculated hydrogen bond energy. The linear correlations found here indicate that the DNA oligomers with 5-substituted-2′-deoxyuridine provide a good model to study the nature of the hydrogen bond in Watson–Crick-type base pairs. Copyright © 2001 John Wiley & Sons, Ltd.
- Published
- 2001
57. [Untitled]
- Author
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Matthias Ernst, Edme H. Hardy, Saburo Aimoto, Toru Kawakami, Masatsune Kainosho, Beat H. Meier, and Andreas Detken
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Series (mathematics) ,Carbon-13 ,Resonance ,Biochemistry ,Molecular physics ,Homonuclear molecule ,chemistry.chemical_compound ,Antamanide ,chemistry ,Solid-state nuclear magnetic resonance ,Heteronuclear molecule ,Computational chemistry ,Adiabatic process ,Spectroscopy - Abstract
The application of adiabatic polarization-transfer experiments to resonance assignment in solid, uniformly 13C-15N-labelled polypeptides is demonstrated for the cyclic decapeptide antamanide. A homonuclear correlation experiment employing the DREAM sequence for adiabatic dipolar transfer yields a complete assignment of the Cα and aliphatic side-chain 13C resonances to amino acid types. The same information can be obtained from a TOBSY experiment using the recently introduced P91 12 TOBSY sequence, which employs the J couplings as a transfer mechanism. A comparison of the two methods is presented. Except for some aromatic phenylalanine resonances, a complete sequence-specific assignment of the 13C and 15N resonances in antamanide is achieved by a series of selective or broadband adiabatic triple-resonance experiments. Heteronuclear transfer by adiabatic-passage Hartmann–Hahn cross polarization is combined with adiabatic homonuclear transfer by the DREAM and rotational-resonance tickling sequences into two- and three-dimensional experiments. The performance of these experiments is evaluated quantitatively.
- Published
- 2001
58. Reelin molecules assemble together to form a large protein complex, which is inhibited by the function-blocking CR-50 antibody
- Author
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Masatsune Kainosho, Eisaku Katayama, Kazunori Nakajima, Naoko Utsunomiya-Tate, Shin-ichi Tate, Katsuhiko Mikoshiba, and Ken Ichiro Kubo
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Macromolecular Substances ,Cell Adhesion Molecules, Neuronal ,Static Electricity ,Dose-Response Relationship, Immunologic ,Nerve Tissue Proteins ,Plasma protein binding ,Biology ,Transfection ,Models, Biological ,CD49c ,Antibodies ,Epitope ,Cell Line ,Epitopes ,Mice ,chemistry.chemical_compound ,Biopolymers ,Cell Adhesion ,Animals ,Reelin ,Phosphorylation ,Binding site ,Phosphotyrosine ,Cells, Cultured ,Extracellular Matrix Proteins ,Binding Sites ,Multidisciplinary ,Serine Endopeptidases ,Tyrosine phosphorylation ,Biological Sciences ,DAB1 ,Mice, Mutant Strains ,Recombinant Proteins ,Cell biology ,Microscopy, Electron ,Reelin Protein ,Solubility ,nervous system ,chemistry ,Mutation ,biology.protein ,Protein Binding ,Signal Transduction - Abstract
Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both in vitro and in vivo . The Reelin–Reelin interaction clearly is inhibited by the function-blocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment.
- Published
- 2000
59. Solution Structure of an RNA Duplex Including a C−U Base Pair
- Author
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Yoshiyuki Tanaka, Chojiro Kojima, Toshio Yamazaki, Takashi S. Kodama, Kazuhiro Yasuno, Shin Miyashita, Akira (Mei) Ono, Akira (Sho) Ono, Masatsune Kainosho, and Yoshimasa Kyogoku
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Oligoribonucleotides ,Nitrogen Isotopes ,Base pair ,Nucleic acid tertiary structure ,Hydrogen bond ,Chemistry ,Chemical shift ,Molecular Sequence Data ,Temperature ,Hydrogen Bonding ,Cytidine ,Nucleic Acid Denaturation ,Biochemistry ,Nucleic acid secondary structure ,Crystallography ,chemistry.chemical_compound ,Duplex (building) ,Intrinsic termination ,Nucleic Acid Conformation ,Base Pairing ,RNA, Double-Stranded - Abstract
The formation of the C-U base pair in a duplex was observed in solution by means of the temperature profile of (15)N chemical shifts, and the precise geometry of the C-U base pair was also determined by NOE-based structure calculation. From the solution structure of the RNA oligomer, r[CGACUCAGG].r[CCUGCGUCG], it was found that a single C-U mismatch preferred being stacked in the duplex rather than being flipped-out even in solution. Moreover, it adopts an irregular geometry, where the amino nitrogen (N4) of the cytidine and keto-oxygen (O4) of the uridine are within hydrogen-bonding distance, as seen in crystals. To further prove the presence of a hydrogen bond in the C-U pair, we employed a point-labeled cytidine at the exocyclic amino nitrogen of the cytidine in the C-U pair. The temperature profile of its (15)N chemical shift showed a sigmoidal transition curve, indicating the presence of a hydrogen bond in the C-U pair in the duplex.
- Published
- 2000
60. Trends in Structure and Growth of Higher Fullerenes Isomer Structure of C86 and C88
- Author
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Tsuyoshi Minami, Yoko Miyake, Yohji Achiba, Masatsune Kainosho, and K. Kikuchi
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Crystallography ,Fullerene ,Absorption spectroscopy ,Chemistry ,Physics::Atomic and Molecular Clusters ,Analytical chemistry ,Molecular symmetry ,Infrared spectroscopy ,Nuclear magnetic resonance spectroscopy ,Carbon-13 NMR ,Condensed Matter Physics ,High-performance liquid chromatography ,Higher fullerenes - Abstract
The isomer structure and stability of higher fullerenes is one of the most interesting issues which might be closely related with general question how a fullerene cage structure is formed. In the present study, the structures of the higher fullerenes with the sizes of C86 and C88 are described based on UV/Visible/nearIR absorption spectra and 13C NMR measurements in solution. For well-understanding the structure and growth of higher fullerenes, the isomer-free samples were prepared for all isomers associated with the C86, and C88 cages by means of a multiple stage high performance liquid chromatography (HPLC). As a result, five different isomers for C86 and C88 were successfully isolated and characterized. For these five different isomers studied in the present work, 13C NMR measurements in solution straightforwardly gave a conclusion that three of them possess the cage structures with a C2 molecular symmetry and two with a Cs molecular symmetry. The possible specific candidates for these five is...
- Published
- 2000
61. 13C-NMR Relaxation Analysis of Nucleic Acid Structure and Dynamics
- Author
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Chojiro KOJIMA and Masatsune KAINOSHO
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- 2000
62. Direct Observation of Hydrogen Bonding in Biomolecules by NMR
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Chojiro KOJIMA and Masatsune KAINOSHO
- Published
- 2000
63. [Untitled]
- Author
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Masatsune Kainosho, Kiyonori Takegoshi, Kenichi Uchida, Kaoru Nomura, and Takehiko Terao
- Subjects
Dipole ,Crystallography ,Solid-state nuclear magnetic resonance ,Spins ,Residual dipolar coupling ,Chemistry ,Magic angle spinning ,Molecule ,Dihedral angle ,Biochemistry ,Molecular physics ,Spectroscopy ,Homonuclear molecule - Abstract
The complete three-dimensional (3D) structure of a glycylisoleucine (Gly-Ile) molecule was determined by individually measuring six dihedral angles with a frequency-selective homonuclear dipolar recoupling method, R2TR (rotational resonance in the tilted rotating frame), using a powder sample of diluted uniformly 13,15-labeled Gly-Ile. Each dihedral angle was obtained by recoupling a dipolar interaction between three or four bonds distant spins concerned or observing a dipolar correlation 2D powder pattern. The 3D structure of a Gly-Ile molecule was also determined by X-ray crystallography, and a good agreement with the NMR result was obtained. The results demonstrate that the R2TR method in a uniformly labeled powder sample can provide the 3D structure without the need to prepare a lot of selectively labeled samples.
- Published
- 2000
64. [Untitled]
- Author
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Hiroyuki Kaji, Nobuhisa Shimba, Eri Kariya, Shin-ichi Tate, and Masatsune Kainosho
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Hydrogen bond ,Chemical shift ,Wild type ,General Medicine ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Hydrophobic effect ,chemistry.chemical_compound ,Crystallography ,chemistry ,Structural Biology ,Cystatin A ,Amide ,Genetics ,Alpha helix - Abstract
The effect of substituting Pro25, located in the α-helical region of the cystatin A structure, with Ser has been studied. The structures of wild type and P25S cystatin A were determined by multidimensional NMR spectroscopy under comparable conditions. These two structures were virtually identical, and the α-helix between Glu15-Lys30 exists with uninterrupted continuity, with a slight bend at residue 25. In order to characterize the possible substitution effects of Pro25 with Ser on the α-helix, the chemical shifts of the amide nitrogens and protons, the generalized order parameters obtained by the analyses of the 15N-1H relaxation data, the amide proton exchange rates, and the NOE networks among the α-helical and surrounding residues were carefully compared. None of these parameters indicated any significant static or dynamic structural differences between the α-helical regions of the wild-type and P25S cystatin A proteins. We therefore conclude that our previous structure of the wild-type cystatin A, in which the α-helix exhibited a sharp kink at Pro25, must be revised. The asymmetric distribution of hydrophobic interactions between the side-chain residues of the α-helix and the rolled β-sheet surface, as revealed by NOEs, may be responsible for the slight bend of the α-helix in both variants and for the destabilized hydrogen bonding of the α-helical residues that follow Pro25/Ser25, as evidenced by increased amide exchange rates.
- Published
- 2000
65. [Untitled]
- Author
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Masatsune Kainosho, Akira Ono, and Chojiro Kojima
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chemistry.chemical_classification ,Residue (complex analysis) ,Isotope ,Base pair ,Hydrogen bond ,Analytical chemistry ,Biochemistry ,chemistry.chemical_compound ,chemistry ,Atom ,Nucleotide ,Thymidine ,Spectroscopy ,DNA - Abstract
15N-15N scalar coupling constants across base pair hydrogen bonds (2hJNN) were studied using residue- and atom-specifically 15N labeled DNA oligomers. The N3 atom selectively 15N enriched 2′-deoxycytidine and thymidine, and the uniformly 15N enriched 2′-deoxyadenosine and 2′-deoxyguanosine, were chemically prepared and incorporated into two DNA oligomers, d(CGCGAATTCGCG)2 and d(CGCAAAAAGCG)•d(CGCTTTTTGCG). This isotope labeling enabled us to determine the 2hJNN value from the splitting of the 15N 1D spectrum. Additionally, it enabled the determination of 2hJNN in D2O quite easily and highly quantitatively. The temperature and DNA sequence dependence were examined for these oligomers. The sequence dependence was not clear; however, a significant decrease of 2hJNN was observed by elevating the temperature. This temperature dependence was not due to the hydrogen exchange, since the addition of 20 mM NH3 did not change the 2hJNN values. The 2hJNN values in D2O were somewhat smaller than those in H2O. As compared to our 15N 1D method, the quantitative HNN-COSY method gave systematically smaller 2hJNN values in our system, due to the lower 15N fraction of our sample (79 and 88% for dA and the other nucleotides, respectively) and the insufficient power of the 15N RF pulse (B1=6.6 kHz). These systematic differences were recovered by theoretical correction of the 15N isotope fraction contribution, by using the composite 15N 180° pulse in a quantitative HNN-COSY experiment.
- Published
- 2000
66. [Untitled]
- Author
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Roland Riek, Masatsune Kainosho, Akira Ono, Kurt Wüthrich, César Fernández, and Konstantin Pervushin
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Chemistry ,Base pair ,Hydrogen bond ,Stereochemistry ,Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid ,Nuclear magnetic resonance spectroscopy of nucleic acids ,Antennapedia ,Biochemistry ,chemistry.chemical_compound ,Computational chemistry ,Molecule ,Nucleic acid structure ,Spectroscopy ,DNA - Abstract
This paper describes NMR measurements of 15N-15N and 1H-15N scalar couplings across hydrogen bonds in Watson-Crick base pairs, h2J(NN) and h1J(HN), in a 17 kDa Antennapedia homeodomain-DNA complex. A new NMR experiment is introduced which relies on zero-quantum coherence-based transverse relaxation-optimized spectroscopy (ZQ-TROSY) and enables measurements of h1J(HN) couplings in larger molecules. The h2JNN and h1J(HN) couplings open a new avenue for comparative studies of DNA duplexes and other forms of nucleic acids free in solution and in complexes with proteins, drugs or possibly other classes of compounds.
- Published
- 2000
67. Assymetric synthesis of (2S,3R)- and (2S,3S)-[2-13C;3-2H] glutamic acid
- Author
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Masatsune Kainosho, Kosuke Okuma, Seiji Tsuchiya, Akira M. Ono, Makoto Oba, Tsutomu Terauchi, and Kozaburo Nishiyama
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chemistry.chemical_compound ,chemistry ,Organic Chemistry ,Drug Discovery ,chemistry.chemical_element ,Organic chemistry ,Glutamic acid ,Biochemistry ,Medicinal chemistry ,Derivative (chemistry) ,Ruthenium ,Catalysis - Abstract
We have developed a synthetic route for (2 S ,3 R )- and (2 S ,3 S )-[2- 13 C;3- 2 H] glutamic acids with high enantioselectivity. The key reactions in this synthesis are the asymmetric reduction of the 2,3-didehydroornithine derivative using the ( S , S )-Et-DuPHOS-Rh catalyst and the oxidation of the δ-position by ruthenium catalysis.
- Published
- 2009
68. Well-Controlled Polymerization of Phenylacetylenes with Organorhodium(I) Complexes: Mechanism and Structure of the Polyenes
- Author
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Takao Ikariya,†,‡ and, Ryoji Noyori, Akira Ono, Masatsune Kainosho, Peter Eckerle, Yasuhisa Kishimoto, and Tatsuya Miyatake
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Tetracoordinate ,Stereochemistry ,chemistry.chemical_element ,General Chemistry ,Biochemistry ,Catalysis ,Rhodium ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Stereospecificity ,chemistry ,Phenylacetylene ,Polymerization ,Pyridine ,Polymer chemistry ,Living polymerization ,Triphenylphosphine - Abstract
A tetracoordinate rhodium complex, Rh(C⋮CC6H5)(nbd)[P(C6H5)3] (nbd = 2,5-norbornadiene), combined with 4-(dimethylamino)pyridine (DMAP) is an excellent initiator for the stereospecific living polymerization of phenylacetylene and its m- and p-substituted derivatives. The rhodium initiator can be generated efficiently by dissociation of triphenylphosphine from isolable Rh(C⋮CC6H5)(nbd)[P(C6H5)3]2 or by reacting Rh(CH3)(nbd)[P(C6H5)3]2 or [Rh(OCH3)(nbd)]2/P(C6H5)3 with one equivalent of phenylacetylene. The use of a phenylethynyl group, triphenylphosphine, and NBD ligand attached to the rhodium center is crucial for the well-controlled polymerization of phenylacetylenes. An additive, DMAP, is necessary to attain low polydispersities of the polymer products. An active rhodium(I) complex bearing a growing polymer chain, NBD, and P(C6H5)3 was isolated from a reaction mixture and was characterized by NMR, GC−MS, XPS, and elemental analyses. The isolated active polymer initiates the further polymerization of the...
- Published
- 1999
69. Stereodivergent Synthesis of (2S,3S,4R,5R)- and (2S,3S,4R,5S)-[3,4,5-D3]Proline Depending on the Substituent of the γ-Lactam Ring
- Author
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Masatsune Kainosho, Kozaburo Nishiyama, Makoto Oba, Tsutomu Terauchi, and Akiko Miyakawa
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chemistry.chemical_compound ,chemistry ,Stereochemistry ,Organic Chemistry ,Substituent ,Lactam ,Proline ,Ring (chemistry) - Published
- 1999
70. Quantitative Measurement of Transverse and Longitudinal Cross-Correlation between13C–1H Dipolar Interaction and13C Chemical Shift Anisotropy: Application to a13C-Labeled DNA Duplex
- Author
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Masatsune Kainosho, Akira Ono, Thomas L. James, and Chojiro Kojima
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Nuclear and High Energy Physics ,Time Factors ,Dna duplex ,Nitrogen ,Biophysics ,Biochemistry ,Molecular physics ,Nuclear magnetic resonance ,A-DNA ,Anisotropy ,Nuclear Magnetic Resonance, Biomolecular ,Carbon Isotopes ,Cross-correlation ,Chemistry ,Temperature ,Spectral density ,Hydrogen Bonding ,DNA ,Condensed Matter Physics ,Transverse plane ,Dipole ,Models, Chemical ,Duplex (building) ,Nucleic Acid Conformation ,Spin Labels - Abstract
Measurement of both longitudinal and transverse relaxation interference (cross-correlation) between 13 C chemical shift anisotropy and 13 C– 1 H dipolar interactions is described. The ratio of the transverse to longitudinal cross-correlation rates readily yields the ratio of spectral densities J (0)/ J (ω C ), independent of any structural attributes such as internuclear distance or chemical shift tensor. The spectral density at zero frequency J (0) is also independent of chemical exchange effects. With limited internal motions, the ratio also enables an accurate evaluation of the correlation time for overall molecular tumbling. Applicability of this approach to investigating dynamics has been demonstrated by measurements made at three temperatures using a DNA decamer duplex with purines randomly enriched to 15% in 13 C.
- Published
- 1999
71. NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ
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Toshimasa Yamazaki, Masayori Inouye, Akira Ono, Ling Qin, Dingjiang Liu, Mitsuhiko Ikura, Masatsune Kainosho, Mark B. Swindells, Kit I. Tong, Chieri Tomomori, Heiyoung Park, Rinku Dutta, Soumitra K. Saha, Rieko Ishima, and Toshiyuki Tanaka
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Multidisciplinary ,Histidine Kinase ,Protein Conformation ,Chemistry ,Escherichia coli Proteins ,Molecular Sequence Data ,Histidine kinase ,Autophosphorylation ,Crystallography, X-Ray ,Recombinant Proteins ,Transmembrane protein ,Two-component regulatory system ,Response regulator ,Biochemistry ,Multienzyme Complexes ,Catalytic Domain ,Escherichia coli ,Phosphorylation ,Amino Acid Sequence ,Protein kinase A ,Protein Kinases ,Histidine ,Bacterial Outer Membrane Proteins - Abstract
Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology1. The histidine–aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes2,3. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region2. The cytoplasmic region contains two functional domains4: domain A (residues 223–289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290–450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heat-shock protein 90 and DNA gyrase B.
- Published
- 1998
72. NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: another example of an ancestral βγ-crystallin precursor structure 1 1Edited by P. E. Wright
- Author
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Sailaja S. Seeram, Masatsune Kainosho, Ayako Ohno, Shin-ichi Tate, Kazumi Hiraga, Kohei Oda, and Mark B. Swindells
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biology ,Structural similarity ,Chemistry ,Dihedral angle ,biology.organism_classification ,Antiparallel (biochemistry) ,Streptomyces ,Crystallography ,Protein structure ,Structural Biology ,Thermolysin ,Crystallin ,Molecular Biology ,Streptomyces nigrescens - Abstract
The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Cα, C′) r.m.s.d. values of 0.45(±0.11) A and 0.57(±0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two β-sheets, each consisting of four antiparallel β-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key β-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain βγ-crystallin proteins, because of its structural similarity to each domain of the βγ-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the βγ-crystallin superfamily are believed to have evolved.
- Published
- 1998
73. Elucidation of the mode of interaction of thermolysin with a proteinaceous metalloproteinase inhibitor, SMPI, based on a model complex structure and a structural dynamics analysis
- Author
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Masatsune Kainosho, Shin-ichi Tate, Sailaja S. Seeram, Kazumi Hiraga, Ayako Ohno, and Kohei Oda
- Subjects
Models, Molecular ,Metalloproteinase ,Binding Sites ,Macromolecular Substances ,Chemistry ,Stereochemistry ,Thermolysin ,Crystal structure ,Matrix metalloproteinase ,Streptomyces ,Crystallography ,Scissile bond ,Bacterial Proteins ,Structural Biology ,Thermodynamics ,Protease Inhibitors ,Serine Proteinase Inhibitors ,Metalloproteinase inhibitor ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Spin relaxation - Abstract
SMPI is a proteinaceous microbial metalloproteinase inhibitor that was isolated from Streptomyces nigrescens TK-23 in 1979. SMPI is known to selectively inhibit the metalloproteinases in the gluzincin family, according to the Rawling and Barrett classification. There has been no report on the interaction of a metalloproteinase in the family of gluzincins with its specific proteinaceous inhibitor. We have solved the solution structure of SMPI by NMR. Here, we report the binding mode of SMPI to thermolysin, based on the model complex structure generated using our high-resolution NMR structure of SMPI and the crystal structure of thermolysin. The obtained complex model shows that the extruded loop of SMPI, with the scissile bond Cys64-Val65, is complementary in shape to the active cleft of thermolysin. In the complex, the Cys64 (P1) carbonyl oxygen atom can form a tetrahedral coordination to the active zinc in thermolysin, and simultaneously, the methyl groups of Val65 (P1′) are closely located in the hydrophobic S1′ pocket in thermolysin. From the electrostatic potential surface calculation, the active loop of SMPI and the active cleft in thermolysin have been shown to be complementary in the surface charge distribution, resulting in the stabilization of the complex. The apparently large active loop is less flexible, but maintains a conformation in the nano- to picosecond time-scale, as elucidated from the 15 N spin relaxation analysis. This is a quite different structural feature of SMPI from the flexible binding loop generally found in the serine proteinase inhibitors, such as SSI and eglin c, and can be related to the narrow specificity of SMPI. The present study provides the first insight into the interaction between a proteinaceous inhibitor and a gluzincin metalloproteinase.
- Published
- 1998
74. Collision-Induced Dissociation Spectra Obtained by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Using a 13C,15N-Doubly Depleted Protein
- Author
-
Satoko Akashi, Koji Takio, Masatsune Kainosho, Hiroshi Matsui, and Shin-ichi Tate
- Subjects
Carbon Isotopes ,Nitrogen Isotopes ,Collision-induced dissociation ,Chemistry ,Molecular Sequence Data ,Analytical chemistry ,Top-down proteomics ,Mass spectrometry ,Tandem mass spectrometry ,Cystatins ,Fourier transform ion cyclotron resonance ,Analytical Chemistry ,Fragmentation (mass spectrometry) ,Spectroscopy, Fourier Transform Infrared ,Mass spectrum ,Amino Acid Sequence ,Ion cyclotron resonance - Abstract
Fourier transform ion cyclotron resonance mass spectra of 13C,15N-doubly depleted cystatin A M65L, produced by Escherichia coli grown on 99.9% [12C]glucose and 99.99% [14N]ammonium sulfate, showed salient monoisotopic peaks composed of 12C and 14N. Collision-induced dissociation spectra were obtained by increasing the capillary-skimmer potential for the electrospray ionization and by extending the trapping time in a radio frequency-only hexapole ion guide. Fragment ions in the spectra could be readily assigned to the amino acid sequence, owing to their markedly improved resolution and sensitivity as compared to those with the natural isotopic composition. Detailed analyses of the fragmentation patterns, facilitated by the use of 13C,15N-doubly depleted proteins, enabled the assignment of approximately 180 fragment ions to the sequence, while natural isotopic cystatin A allowed the assignment of approximately 110 fragment ions. Interestingly, no fragmentation was detected between residues 50-61 and 62-67, which are stretches known to be involved in the antiparallel beta-sheet at the center of the protein.
- Published
- 1998
75. Determination of peptide φ angles in solids by relayed anisotropy correlation NMR
- Author
-
Takehiko Terao, Makoto Oba, Yoshitaka Ishii, Masatsune Kainosho, Tsutomu Terauchi, Kozaburo Nishiyama, and Koichi Hirao
- Subjects
chemistry.chemical_classification ,Nuclear and High Energy Physics ,Radiation ,Peptide ,General Chemistry ,Dihedral angle ,Polarization (waves) ,Dipole ,Crystallography ,Solid-state nuclear magnetic resonance ,chemistry ,Present method ,Magic angle spinning ,Anisotropy ,Humans ,Peptides ,Nuclear Magnetic Resonance, Biomolecular ,Instrumentation - Abstract
A solid state NMR method is presented for determination of a backbone dihedral angle phi in peptides, being based on the previously reported method, relayed anisotropy correlation (RACO) NMR [Y. Ishii et al., Chem. Phys. Lett. 256 (1996) 133]. In the present method, the 15N-1H and the 13C-1H dipolar tensors in the 1H-15N-13C-1H system are two-dimensionally (2D) correlated via polarization transfer from 15N to 13C under magic angle spinning (MAS). This method was applied to N-acetyl[1,2-13C,15N]D,L-valine, and the H-C-N-H dihedral angle was determined to be 154.0 +/- 1.4 degrees or 206.0 +/- 1.4 degrees, the former agreeing with the X-ray value of 154 +/- 5 degrees.
- Published
- 1998
76. Synthesis of [5′-2H1]-nucleosides with defined (5′S)/(5′R) - ratios
- Author
-
Yu Oogo, Akira (Mei) Ono, Akira Ono, and Masatsune Kainosho
- Subjects
chemistry.chemical_classification ,Reaction conditions ,chemistry ,Oligonucleotide ,Stereochemistry ,Organic Chemistry ,Drug Discovery ,Pentose ,Organic chemistry ,Stereoselectivity ,Biochemistry - Abstract
A method for preparing 5′-monodeuterated nucleosides with defined (5′S)(5′R)-ratios, starting from glucose, is described. The 5-oxopentose derivatives, derived from glucose, were treated with LiAlD4 in the presence of LiI and/or t-amylalcohol to give stereoselectively 5-monodeuterated pentose derivatives. Using various reaction conditions, the stereoselectivity of the monodeuterated 5-hydroxymethyl group could be partially controlled, in the range of isotopomeric ratios, (5S)(5R), of 4: 1 to 1:7.4. Using appropriate mixtures of the 5-monodeuterated pentoses with different isotopomeric ratios, we could prepare 5′-monodeuterated nucleosides with defined isotopomeric ratios, which will be useful for NMR studies of oligonucleotides.
- Published
- 1998
77. Systematic synthesis of specifically 13C/2H - labeled nucleosides from [ul-13C6]-d-glucose
- Author
-
Akira (Mei) Ono, Toko Shiina, Akira Ono, and Masatsune Kainosho
- Subjects
chemistry.chemical_compound ,Heteronuclear molecule ,Deuterium ,Stereochemistry ,Chemistry ,D-Glucose ,Organic Chemistry ,Drug Discovery ,Nucleic acid ,Nuclear magnetic resonance spectroscopy ,Systematic synthesis ,Biochemistry - Abstract
A systematic approach, which combines previously reported reactions with appropriate modifications, was established for preparing a variety of selectively deuterated nucleosides from glucose. We have developed a systematic method of synthesizing specifically deuterated nucleosides from glucose. Selectively 13C/2H - doubly labeled nucleosides, such as [1′,2′,3′,4′,5′-13C5;3′,5′-2H2]- and [1′,2′,3′,4′,5′-13C5;4′,5′-2H2]-adenosines, have been synthesized starting from [ul-13C6]- d -glucose. These labeled nucleosides are very useful for precise conformational analyses of nucleic acids in solution by heteronuclear multidimensional NMR spectroscopy.
- Published
- 1998
78. [Untitled]
- Author
-
Masatsune Kainosho, Yoshiharu Ishido, Etsuko Kawashima, Chojiro Kojima, Yoshimasa Kyogoku, Kenshiro Ohshima, and Keizo Toyama
- Subjects
chemistry.chemical_classification ,Coupling constant ,Geminal ,Stereochemistry ,Ring (chemistry) ,Biochemistry ,chemistry.chemical_compound ,Stereospecificity ,Deuterium ,chemistry ,Nucleotide ,Methylene ,Two-dimensional nuclear magnetic resonance spectroscopy ,Spectroscopy - Abstract
The stereoselective deuterium labeling at the 5' methylene protons of the ribose ring recently developed by Kawashima et al. [1995, Tetrahedron Lett., 36, 6699–6700] enabled the assignment of pro-R and pro-S protons at the 5' position. The deuterium-labeled nucleotides, [(5'S)-2H]- and [(5'R)-2H]-diastereomers, in an approximate ratio of 2:1, were incorporated in the decamer 5'-d(GCATTAATGC)-3'. Thus, both pro-R and pro-S methylene proton signals without geminal coupling appeared in the NOESY and DQF-COSY spectra. Complete stereospecific assignments and simplified spin systems enabled the determination of 15 3J coupling constants between H4' and H5'/H5", and the unambiguous assignment of 135 NOESY cross peaks originating from H4'/H5'/H5" resonances.
- Published
- 1998
79. [Untitled]
- Author
-
Mitsuhiko Ikura, Lubert Stryer, Toshiyuki Tanaka, Masatsune Kainosho, and James B. Ames
- Subjects
Crystallography ,Heteronuclear molecule ,Hydrogen bond ,Valine ,Covalent bond ,Chemistry ,lipids (amino acids, peptides, and proteins) ,Nuclear magnetic resonance spectroscopy ,Dihedral angle ,Biochemistry ,Protein secondary structure ,Spectroscopy ,Myristoylation - Abstract
The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca 2C -free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR techniques. In some experiments, the myristoyl group covalently attached to the protein N-terminus was labeled with 13 C and the protein was unlabeled or vice versa; in others, both were 13 C-labeled. This differential labeling strategy was essential for structural refinement and can be applied to other acylated proteins. Stereospecific assignments of 41 pairs of b-methylene protons and 48 methyl groups of valine and leucine were included in the structure refinement. The refined structure was constructed using a total of 3679 experimental NMR restraints, comprising 3242 approximate interproton distance restraints (including 153 between the myristoyl group and the polypeptide), 140 distance restraints for 70 backbone hydrogen bonds, and 297 torsion angle restraints. The atomic rms deviations about the averaged minimized coordinate positions for the secondary structure region of the N-terminal and C-terminal domains are 0.44 0.07 and 0.55 0.18 A for backbone atoms, and 1.09 0.07 and 1.10 0.15 A for all heavy atoms, respectively. The refined structure allows for a detailed analysis of the myristoyl binding pocket. The myristoyl group is in a slightly bent conformation: the average distance between C1 and C14 atoms of the myristoyl group is 14.6 A. Hydrophobic residues Leu 28 ,T rp 31 ,a nd Tyr 32 form a cluster that interacts with the front end of the myristoyl group (C1‐C8), whereas residues Phe 49 ,P he 56 ,T yr 86 ,V al 87 ,a nd Leu 90 interact with the tail end (C9‐C14). The relatively deep hydrophobic pocket that binds the myristoyl group (C14:0) could also accommodate other naturally occurring acyl groups such as C12:0, C14:1, and C14:2 chains.
- Published
- 1998
80. [Untitled]
- Author
-
Shin-ichi Tate, Akira Ono, Hideo Iwaï, Masatsune Kainosho, Thomas Szyperski, César Fernández, and Kurt Wüthrich
- Subjects
chemistry.chemical_compound ,Crystallography ,Deoxyribose ,Duplex (building) ,Chemistry ,Chemical shift ,Nucleic acid ,Protein-DNA complex ,Antennapedia ,Biochemistry ,Two-dimensional nuclear magnetic resonance spectroscopy ,Spectroscopy ,DNA - Abstract
Nearly complete 1H, 13C and15 N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly 15N-labeled Antennapedia homeodomain. The DNA was either fully 13C,15N-labeled or contained uniformly 13C, 15N-labeled nucleotides only at those positions which form the protein–DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain–DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via1 H–1H NOEs observed in 3D13 C-resolved NOESY; and (iii) assignment of the imino and amino groups via 1H–1H NOEs and15 N–1H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein–DNA complex was greatly facilitated by the fact that 1H signals of the protein were filtered out in 13C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding 13C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.
- Published
- 1998
81. Protein Structure Dynamics Elucidated through Spin-Relaxation Analysis by NMR
- Author
-
Shin-ichi Tate and Masatsune Kainosho
- Subjects
Materials science ,Protein structure ,Chemical physics ,Dynamics (mechanics) ,Spin relaxation - Published
- 1998
82. [Untitled]
- Author
-
Koji Takio, Jonathan A. Cooper, Naoshi Dohmae, Ernest D. Laue, Masatsune Kainosho, Shigeyuki Yokoyama, Takanori Kigawa, Tohru Terada, Takashi Yabuki, and Yutaka Ito
- Subjects
chemistry.chemical_classification ,Cell-free protein synthesis ,medicine.disease_cause ,Biochemistry ,Amino acid ,chemistry.chemical_compound ,chemistry ,medicine ,Protein biosynthesis ,Threonine ,Tyrosine ,Escherichia coli ,Spectroscopy ,DNA ,Heteronuclear single quantum coherence spectroscopy - Abstract
We developed two methods for stable-isotope labeling of proteins by cell-free synthesis. Firstly, we applied cell-free synthesis to the dual amino acid-selective 13C-15N labeling method, originally developed for in vivo systems by Kainosho and co-workers. For this purpose, we took one of the advantages of a cell-free protein synthesis system; the amino acid-selective stable-isotope labeling is free of the isotope scrambling problem. The targets of selective observation were Thr35 and Ser39 in the ‘effector region’ (residues 32–40) of the Ras protein complexed with the Ras-binding domain of c-Raf-1 (Raf RBD) (the total molecular mass is about 30 kDa). Using a 15-mL Escherichia coli cell-free system, which was optimized to produce about 0.4 mg of Ras protein per 1-mL reaction, with 2 mg each of DL-[13C′]proline and L-[15N]threonine, we obtained about 6 mg of Ras protein. As the Pro–Thr sequence is unique in the Ras protein, the Thr35 cross peak of the Ras•Raf RBD complex was unambiguously identified by the 2D 1H–15N HNCO experiment. The Ser39 cross peak was similarly identified with the [13C′]Asp/[15N]Ser-selectively labeled Ras protein. There were no isotope scrambling problems in this study. Secondly, we have established a method for producing a milligram quantity of site-specifically stable-isotope labeled protein by a cell-free system involving amber suppression. The E. coli amber suppressor tRNATyr_CUA (25 mg) was prepared by in vitro transcription with T7 RNA polymerase. We aminoacylated the tRNATyr_CUA transcript with purified E. coli tyrosyl-tRNA synthetase, using 2 mg of l-[15N]tyrosine. In the gene encoding the Ras protein, the codon for Tyr32 was changed to an amber codon (TAG). This template DNA and the [15N]Tyr-tRNATyr_CUA were reacted for 30 min in 30 mL of E. coli cell-free system. The subsequent purification yielded 2.2 mg of [15N]Tyr32-Ras protein. In the 1H–15N HSQC spectrum of the labeled Ras protein, only one cross peak was observed, which was unambiguously assigned to Tyr32.
- Published
- 1998
83. Novel approach to diastereoselective synthesis of 2?-deoxy[5?-2H1]ribonucleoside derivatives by reduction of the corresponding 5?-O-acetyl-2?-deoxy-5?-phenylselenoribonucleoside derivatives with a Bu3Sn2H-Et3B system
- Author
-
Yoshimasa Kyogoku, Etsuko Kawashima, Keizo Toyama, Masatsune Kainosho, Kenshiro Ohshima, and Yoshiharu Ishido
- Subjects
Pharmacology ,Chemistry ,Stereochemistry ,Organic Chemistry ,Guanosine ,Cytidine ,Ribonucleoside ,Catalysis ,Analytical Chemistry ,chemistry.chemical_compound ,Drug Discovery ,Chirality (chemistry) ,Thymidine ,Spectroscopy - Abstract
Diastereoselective syntheses of [5′-2H]thymidine (6-T) [(5′R)/(5′S) = 31:69], N4-benzoyl-2′-deoxy[5′-2H]cytidine (6-CBz) [(5′R)/(5′S) = 27:76], N6-benzoyl-2′-deoxy[5′-2H]adenosine (6-ABz [(5′R)/(5′S) = 39:61], and 2′-deoxy-N2-isobutyryl[5′-2H]guanosine (6-GiBu) [(5′R)/(5′S) = 20:80] were attained by a radical reductive deuteration reaction of the corresponding 5′-O-acetyl-3′-O-TBDMS-2′-deoxy-5′-phenylselenoribonucleosides (4) with a Bu3Sn2H-Et3B system at
- Published
- 1997
84. Synthesis of (5′S)-[5′-2H1;1′,2′,3′,4′,5′-13C5]-Thymidine via stereoselective deuteration of a 5-oxoribose derivative
- Author
-
Akira (Mei) Ono, Akira Ono, and Masatsune Kainosho
- Subjects
Chemistry ,Stereochemistry ,Organic Chemistry ,Biochemistry ,chemistry.chemical_compound ,Stereospecificity ,Bromide ,Yield (chemistry) ,Drug Discovery ,Nucleic acid ,Stereoselectivity ,Methylene ,Thymidine ,Derivative (chemistry) - Abstract
(5′S)-[5′-2H1;1′,2′,3′,4′,5′-13C5]-Thymidine has been synthesized by a stereoselective deuteride transfer reaction from (−)- or (+)-[2-2H1]-isobornyloxymagnesium bromide to a 5-oxoribose derivative, which can be readily prepared from [13C6]- d -glucose. The overall yield from d -glucose to thymidine was 27%. The various nucleosides with a stereoselective 2H-label together with 13C at the C5′ position, which have become available by the present method, will be quite useful for stereospecific assignment of the diastereotopic C5′ methylene signals, and also for conformational analyses of the O5′C5′ bonds in nucleic acid oligomers.
- Published
- 1997
85. Use of H/D isotope effects to gather information about hydrogen bonding and hydrogen exchange rates
- Author
-
Mitsuhiro Takeda, Yohei Miyanoiri, Tsutomu Terauchi, Masatsune Kainosho, and Chun Jiun Yang
- Subjects
Models, Molecular ,Nuclear and High Energy Physics ,Hydrogen ,Protein Conformation ,Biophysics ,chemistry.chemical_element ,Too quickly ,Biochemistry ,Computational chemistry ,Kinetic isotope effect ,Deuterium Oxide ,Nuclear Magnetic Resonance, Biomolecular ,Hydrogen exchange ,Carbon Isotopes ,Isotope ,Hydrogen bond ,Deuterium Exchange Measurement ,Proteins ,Hydrogen Bonding ,Carbon-13 NMR ,Condensed Matter Physics ,Deuterium ,chemistry ,Polar ,Atomic physics ,Protons - Abstract
Polar side-chains in proteins play important roles in forming and maintaining three-dimensional structures, and thus participate in various biological functions. Until recently, most protein NMR studies have focused on the non-exchangeable protons of amino acid residues. The exchangeable protons attached to polar groups, such as hydroxyl (OH), sulfhydryl (SH), and amino (NH2) groups, have mostly been ignored, because in many cases these hydrogen atoms exchange too quickly with water protons, making NMR observations impractical. However, in certain environments, such as deep within the hydrophobic interior of a protein, or in a strong hydrogen bond to other polar groups or interacting ligands, the protons attached to polar groups may exhibit slow hydrogen exchange rates and thus become NMR accessible. To explore the structural and biological implications of the interactions involving polar side-chains, we have developed versatile NMR methods to detect such cases by observing the line shapes of (13)C NMR signals near the polar groups, which are affected by deuterium-proton isotope shifts in a mixture of H2O and D2O. These methods allow the detection of polar side-chains with slow hydrogen-deuterium exchange rates, and therefore provide opportunities to retrieve information about the polar side-chains, which might otherwise be overlooked by conventional NMR experiments. Future prospects of applications using deuterium-proton isotope shifts to retrieve missing structural and dynamic information of proteins are discussed.
- Published
- 2013
86. Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids
- Author
-
Tsutomu Terauchi, Mitsuhiro Takeda, Kosuke Okuma, Akira M. Ono, Masatsune Kainosho, and Yohei Miyanoiri
- Subjects
Stereochemistry ,Gene Expression ,medicine.disease_cause ,Biochemistry ,Hemiterpenes ,Valine ,Leucine ,Labelling ,Malate synthase ,medicine ,Escherichia coli ,Amino Acids ,Gene ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,chemistry.chemical_classification ,Carbon Isotopes ,biology ,Escherichia coli Proteins ,Carbon-13 ,Keto Acids ,Amino acid ,chemistry ,Isotope Labeling ,biology.protein - Abstract
The (1)H-(13)C HMQC signals of the (13)CH3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically (13)CH3-labeled [U-(2)H;(15)N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.
- Published
- 2013
87. Stereo-Array Isotope Labeling (SAIL) Method
- Author
-
Masatsune Kainosho
- Published
- 2013
88. Evolution and diversification of the plant gibberellin receptor GID1.
- Author
-
Hideki Yoshida, Eiichi Tanimoto, Takaaki Hirai, Yohei Miyanoiri, Rie Mitani, Mayuko Kawamura, Mitsuhiro Takeda, Sayaka Takehara, Ko Hirano, Masatsune Kainosho, Takashi Akagi, Makoto Matsuoka, and Miyako Ueguchi-Tanaka
- Subjects
GIBBERELLINS ,PLANT hormones ,CARBOXYLESTERASES ,ARABIDOPSIS ,SOYBEAN ,LETTUCE - Abstract
The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA
4 , new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution. [ABSTRACT FROM AUTHOR]- Published
- 2018
- Full Text
- View/download PDF
89. C5′ Methylene Proton Signal Assignment of DNA/RNA Oligomers Labeled with C5′-Monodeuterated Nucleosides by1H-31P HSQC Spectroscopy
- Author
-
Shin-ichi Tate, Etsuko Kawashima, Yoshiharu Ishido, Akira Ono, Takako Makita, and Masatsune Kainosho
- Subjects
Proton ,Analytical chemistry ,General Chemistry ,Oligomer ,chemistry.chemical_compound ,Crystallography ,chemistry ,Deuterium ,Proton NMR ,General Materials Science ,A-DNA ,Methylene ,Heteronuclear single quantum coherence spectroscopy ,Magnetic dipole–dipole interaction - Abstract
A new strategy for assigning the methylene protons attached to the C5′ of nucleic acids is described for a DNA dodecamer duplex, d(CGCGAATTCGCG)2. It employs 2D 1H–31P HSQC spectroscopy for DNA composed of deoxyribonucleotides, having one deuteron at their C5′ positions. Due to the lack of scalar as well as dipolar coupling between the C5′ methylene proton pair, the cross peaks for the residual H5′/H5″ protons and the 31P nuclei (at the 5′-terminal side) appear as much narrower signals, as compared to those of the intact proton pairs in the unlabeled dodecamer. The overall resolution and sensitivity of the 1H–31P HSQC cross peaks for the residual signals of the deuterated C5′-methylenes have thus been drastically improved over those for the unlabeled oligomer. All of the H5′/H5″ signals of the dodecamer are clearly observed, except for the unphosphorylated 5′-terminus, and each has been assigned. Most of the diastereotopic proton pairs at C5′ showed different relative peak intensities, which may be related to the fact that the present C5′ monodeuteration procedures proceed slightly stereoselectively, namely 60–70% 2H at H5′ (pro-S) and 40–30% 2H at H5″ (pro-R), respectively. The possibility of correlating these peak intensity difference with the stereospecific assignment is discussed.
- Published
- 1996
90. Relayed anisotropy correlation NMR: determination of dihedral angles in solids
- Author
-
Masatsune Kainosho, Takehiko Terao, and Yoshitaka Ishii
- Subjects
Crystallography ,Mutual orientation ,Chemistry ,Group (periodic table) ,Orientation (geometry) ,General Physics and Astronomy ,Tensor ,Physical and Theoretical Chemistry ,Dihedral angle ,Anisotropy ,Polarization (waves) ,Magnetic dipole–dipole interaction - Abstract
A two-dimensional solid-state NMR method is proposed for determining the mutual orientation of the two interaction tensors in each of which a different chemical group participates, from which information on the dihedral angle can be extracted. The two-dimensional powder pattern was observed for 1,2- 13 C-labeled DL-alanine as a demonstration, correlating the 13 C 1 chemical shift anisotropy and the 13 C 2 1 H dipolar coupling via polarization transfer. The OC 1 C 2 H dihedral angle as well as the orientation of the 13 C 1 chemical shift tensor were determined from the spectrum.
- Published
- 1996
91. Significance of the Highly Conserved Gly-4 Residue in Human Cystatin A1
- Author
-
Yukihito Ohyama, Shin-ichi Tate, Masatsune Kainosho, Hiroyuki Kaji, Tatsuya Samejima, Kazunori Shibuya, and Fuyuhiko Inagaki
- Subjects
chemistry.chemical_classification ,Wild type ,General Medicine ,medicine.disease_cause ,Biochemistry ,Cassette mutagenesis ,Amino acid ,Papain ,chemistry.chemical_compound ,chemistry ,Cystatin A ,medicine ,Cyanogen bromide ,Cystatin ,Molecular Biology ,Escherichia coli - Abstract
The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A (2-98)) was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Ctst A(2-98)), called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A(2-98)[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory effect towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.
- Published
- 1995
92. Determination of heteronuclear three-bond J-coupling constants in peptides by a simple heteronuclear relayed E.COSY experiment
- Author
-
Richard R. Ernst, Saburo Aimoto, Jürgen M. Schmidt, and Masatsune Kainosho
- Subjects
Coupling constant ,Magnetic Resonance Spectroscopy ,Molecular Structure ,Chemistry ,Phenylalanine ,Molecular Sequence Data ,Analytical chemistry ,Valine ,Dihedral angle ,J-coupling ,Peptides, Cyclic ,Biochemistry ,Quantitative determination ,Crystallography ,chemistry.chemical_compound ,Heteronuclear molecule ,Antamanide ,Simple (abstract algebra) ,Amino Acid Sequence ,Peptides ,Spectroscopy - Abstract
A simple heteronuclear relayed E.COSY pulse sequence with a minimum number of pulses is proposed for the quantitative determination of heteronuclear three-bond J-coupling constants in uniformly 13C-enriched polypeptide samples. Numerous heteronuclear three-bond coupling constants, including \({}^3{\text{J}}_{{\text{H}}^{\text{N}} {\text{C}}'} \), \({}^3{\text{J}}_{{\text{H}}^{\text{N}} {\text{C}}^\beta } \), \({}^3{\text{J}}_{{\text{H}}^\beta {\text{C}}'} \), and \({}^3{\text{J}}_{{\text{H}}^\alpha {\text{C}}^\gamma } \), can be determined for each residue from a single heteronuclear relayed E.COSY spectrum. Couplings relevant for stereospecific assignments as well as for the determination of dihedral angles in the amino acid backbone and in side chains are obtained. The method is demonstrated on the uniformly 13C-enriched decapeptide antamanide (-Val1-Pro2-Pro3-Ala4-Phe5-Phe6-Pro7-Pro8-Phe9-Phe10-).
- Published
- 1995
93. Highly Diastereoselective Synthesis of (2'S)-[2'-2H]-2'-Deoxyribonucleosides from the Corresponding Ribonucleosides
- Author
-
Masayoshi Miyahara, Masatsune Kainosho, Yoshiharu Ishido, Mohamed F. Radwan, Etsuko Kawashima, Yukio Aoyama, Takeshi Sekine, and Yoshimasa Kyogoku
- Subjects
chemistry.chemical_compound ,Deoxyribonucleosides ,Chemistry ,Stereochemistry ,Bromide ,Genetics ,Molecular Medicine ,Halogenation ,Stereoselectivity ,General Medicine ,Biochemistry - Abstract
The four (2′S)-[2′-2H]-2′-deoxynucleosides (>90 atom % 2H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB2H4 in EtOH-H2O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu3SnH-Et3B reduction of the resulting bromide, and, finally, unmasking.
- Published
- 1995
94. Synthesis of<scp>L</scp>-threo- and<scp>L</scp>-erythro-[1-13C, 2,3-2H2]amino acids: novel probes for conformational analysis of peptide side chains
- Author
-
Makoto Oba, Kozaburo Nishiyama, Mika Fukuoka, Masatsune Kainosho, and Ryuichi Ueno
- Subjects
chemistry.chemical_classification ,chemistry ,Stereochemistry ,Diastereomer ,Side chain ,Stereoselectivity ,Peptide ,Nuclear magnetic resonance spectroscopy ,Racemization ,Conformational isomerism ,Amino acid - Abstract
An efficient and convenient route for the preparation of L-threo- and L-erythro-[1-l3C, 2,3-2H2]amino acids 5 as probes for the conformational analysis of peptide side chains by NMR spectroscopy is described. Stereoselective incorporation of deuterium into the α,β-positions of amino acid 5 was accomplished by catalytic deuteriation of dehydroamino acid derivatives 1 and 2 followed by a combination of enzymic optical resolution and the racemization at the 2-position. Using the doubly labelled amino acids, it was possible to obtain vicinal coupling constants between carbonyl carbon and prochiral β-protons, J(13C1–1Hβ1) and J(13C1–1Hβ2), through 13C NMR spectroscopy alone. We also demonstrate the determination of the fractional populations of rotamers in respect of the Cα–Cβ bond of the amino acids using the measured coupling constants.
- Published
- 1995
95. Biosynthesis of Lactacystin
- Author
-
Akira Nakagawa, Satoshi Omura, Masatsune Kainosho, Keiichi Matsuzaki, Kenichi Uchida, Yoko Miyake, and Senji Takahashi
- Subjects
Pharmacology ,Magnetic Resonance Spectroscopy ,Molecular Structure ,Streptomycetaceae ,Stereochemistry ,Lactacystin ,Stereoisomerism ,macromolecular substances ,Nuclear magnetic resonance spectroscopy ,Cysteine Proteinase Inhibitors ,Biology ,biology.organism_classification ,Streptomyces ,Acetylcysteine ,Anti-Bacterial Agents ,chemistry.chemical_compound ,chemistry ,Biosynthesis ,Drug Discovery ,Moiety ,Leucine - Abstract
The biosynthesis of lactacystin, a new microbial metabolite which induces differentiation of neuroblastoma cells, was studied by the feeding experiments of various 13C-labeled compounds and NMR spectroscopic analysis. The feeding experiments showed that lactacystin consists of three containing gamma-lactam moiety arises by a condensation between methylmalonic semialdehyde and C alpha position of L-leucine, followed by intramolecular cyclization. Two diastereotopic methyls, C-11 and C-12 of lactacystin were found to originate from the pro-R and pro-S methyls of leucine, respectively, as shown by incorporating a new type of chiral 13C-labeled L-leucine.
- Published
- 1995
96. Methylation Dependent Functional Switch Mechanism of the Escherichia coli Ada Protein
- Author
-
Yoshiko Akitomo, Mutsuo Sekiguchi, Tadayasu Ohkubo, Masatsune Kainosho, Hitoshi Sakashita, Takahiko Sakuma, and Kosuke Morikawa
- Subjects
Mutagenesis ,General Physics and Astronomy ,Helix-turn-helix ,General Medicine ,Methylation ,Biology ,medicine.disease_cause ,DNA methyltransferase ,chemistry.chemical_compound ,Biochemistry ,chemistry ,medicine ,General Agricultural and Biological Sciences ,Site-directed mutagenesis ,Transcription factor ,Escherichia coli ,DNA - Abstract
NMR studies have revealed the mechanism of a functional switch in the Escherichia coli Ada protein, which converts the DNA methyltransferase to a transcriptional regulator. The Ada protein acquires its ability to bind specific DNA sequences after an irreversible methyl transfer to its own cysteine residue (Cys69) from methyl-phosphotriesters within the alkylated DNA. NMR analysis have revealed that the segment from residues 102 to 123 forms a helix-turn-helix structure, and a site-directed mutagenesis study has identified the second helix to play a crucial role in specific recognition of DNA. NMR experiments on the methylated protein-DNA complex showed that although the major contacts are made by residues within the recognition helix, the S-methyl group at Cys69 come into direct contact with the cognate DNA. Therefore, the direct contact of this region after methylation is the “switch” which converts the Ada protein from a nonspecific DNA binding form to a transcription factor.
- Published
- 1995
97. Stereo-array isotope labeling method for studying protein structure and dynamics
- Author
-
Yohei, Miyanoiri, Mitsuhiro, Takeda, and Masatsune, Kainosho
- Subjects
Cell-Free System ,Isotope Labeling ,Proteins ,Disulfides ,Nuclear Magnetic Resonance, Biomolecular - Abstract
The stereo-array isotope labeling (SAIL) method utilizes proteins with isotope labeling patterns optimized with regard to an intended NMR study. The SAIL proteins are prepared by incorporating chemically synthesized amino acids into target proteins, using a cell-free protein synthesis system or a cellular expression system. Over the past decade, the SAIL method has been facilitating a wide variety of new investigations, including high-resolution structure determinations of large proteins and investigations of protein dynamics. In this chapter, the applications of SAIL-related approaches are introduced.
- Published
- 2012
98. Cell-free protein synthesis using E. coli cell extract for NMR studies
- Author
-
Mitsuhiro, Takeda and Masatsune, Kainosho
- Subjects
Cell-Free System ,Protein Biosynthesis ,Escherichia coli ,Nuclear Magnetic Resonance, Biomolecular - Abstract
The use of cell-free protein production systems for producing isotope labeled proteins generates new opportunities to perform unprecedented NMR studies. As compared with conventional cellular expression systems, the scrambling and dilution of amino acids are highly suppressed in the cell-free reaction, allowing the production of proteins with a wide variety of residue and site-specific isotope labeling patterns. In this chapter, the procedure for cell-free protein synthesis for NMR studies, using an E. coli extract, is introduced.
- Published
- 2012
99. Kainosho, Masatsune: Developing Stable-Isotope- Aided NMR Technologies for Biological Systems
- Author
-
Masatsune Kainosho
- Subjects
Chemistry ,Stable isotope ratio ,Subtilisin ,Organic chemistry - Abstract
A personal recollection is provided on my research, aiming to develop various stable-isotope aided NMR methods for amino acids, peptides, and proteins. A salient trait of our approaches is to extensively use NMR samples composed of site – and stereospecifically isotope-labeled components, which can be prepared by microbial fermentations, enzyme-catalyzed reactions, and chiral organic syntheses – all areas in which Japan leads the world. The selective 13C, 15N-double labeling method and the stereo-array isotope labeling (SAIL) method have been chosen as representatives of my early and latest work, respectively, to illustrate the features of our approaches. Keywords: isotope-aided NMR methods; selective 13C, 15N-double labeling; Streptomyces subtilisin inhibitor (S-SI); stereospecific deuteration; stereo-array isotope labeling (SAIL) method
- Published
- 2012
100. Cell-Free Protein Synthesis Using E. coli Cell Extract for NMR Studies
- Author
-
Masatsune Kainosho and Mitsuhiro Takeda
- Subjects
chemistry.chemical_classification ,Residue (chemistry) ,Cell-free protein synthesis ,medicine.anatomical_structure ,chemistry ,Isotope ,Biochemistry ,Cell ,medicine ,Protein biosynthesis ,Amino acid - Abstract
The use of cell-free protein production systems for producing isotope labeled proteins generates new opportunities to perform unprecedented NMR studies. As compared with conventional cellular expression systems, the scrambling and dilution of amino acids are highly suppressed in the cell-free reaction, allowing the production of proteins with a wide variety of residue and site-specific isotope labeling patterns. In this chapter, the procedure for cell-free protein synthesis for NMR studies, using an E. coli extract, is introduced.
- Published
- 2012
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