253 results on '"Masatsune Kainosho"'
Search Results
2. Chemical Conformation of the Essential Glutamate Site of the
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Yasuto, Todokoro, Su-Jin, Kang, Toshiharu, Suzuki, Takahisa, Ikegami, Masatsune, Kainosho, Masasuke, Yoshida, Toshimichi, Fujiwara, and Hideo, Akutsu
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Protein Subunits ,Proton-Translocating ATPases ,Adenosine Triphosphate ,Protein Conformation ,Cryoelectron Microscopy ,Glutamic Acid ,Bacillus ,Protons - Abstract
Proton translocation through the membrane-embedded F
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- 2022
3. Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method
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Masatsune Kainosho, Tsutomu Terauchi, Yohei Miyanoiri, and Mitsuhiro Takeda
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QC501-766 ,010405 organic chemistry ,Chemistry ,Stereochemistry ,Chemical shift ,Protonation ,Aromaticity ,Nuclear magnetic resonance spectroscopy ,Carbon-13 NMR ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Electricity and magnetism ,chemistry.chemical_compound ,Deprotonation ,TheoryofComputation_MATHEMATICALLOGICANDFORMALLANGUAGES ,Side chain ,Methylene - Abstract
Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated using NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of staphylococcal nuclease (SNase), which contains 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of ∼ 5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereospecifically assigned using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys. The complete set of assigned 1H, 13C, and 15N NMR signals for the Lys side-chain moieties affords useful structural information. For example, the set includes the characteristic chemical shifts for the 13Cδ, 13Cε, and 15Nζ signals for Lys-66, which has the deprotonated ζ-amino group, and the large upfield shifts for the 1H and 13C signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are indicative of nearby aromatic rings. The 13Cε and 15Nζ chemical shifts of the SNase variant selectively labeled with either [ε-13C;ε,ε-D2]-Lys or SAIL-Lys, dissolved in H2O and D2O, showed that the deuterium-induced shifts for Lys-66 were substantially different from those of the other 20 Lys residues. Namely, the deuterium-induced shifts of the 13Cε and 15Nζ signals depend on the ionization states of the ζ-amino group, i.e., −0.32 ppm for Δδ13Cε [NζD3+-NζH3+] vs. −0.21 ppm for Δδ13Cε [NζD2-NζH2] and −1.1 ppm for Δδ15Nζ[NζD3+-NζH3+] vs. −1.8 ppm for Δδ15Nζ[NζD2-NζH2]. Since the 1D 13C NMR spectrum of a protein selectively labeled with [ε-13C;ε,ε-D2]-Lys shows narrow (> 2 Hz) and well-dispersed 13C signals, the deuterium-induced shift difference of 0.11 ppm for the protonated and deprotonated ζ-amino groups, which corresponds to 16.5 Hz at a field strength of 14 T (150 MHz for 13C), could be accurately measured. Although the isotope shift difference itself may not be absolutely decisive to distinguish the ionization state of the ζ-amino group, the 13Cδ, 13Cε, and 15Nζ signals for a Lys residue with a deprotonated ζ-amino group are likely to exhibit distinctive chemical shifts as compared to the normal residues with protonated ζ-amino groups. Therefore, the isotope shifts would provide a useful auxiliary index for identifying Lys residues with deprotonated ζ-amino groups at physiological pH levels.
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- 2021
4. Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific 1H–13C Labeling and Fast Magic-Angle Spinning NMR
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Diego F. Gauto, David Gajan, Roman J. Lichtenecker, Alessandro Barducci, Audrey Hessel, Masatsune Kainosho, Paul Schanda, Pavel Macek, Yohei Miyanoiri, Tsutomu Terauchi, Jérôme Boisbouvier, and Hugo Pacheco de Freitas Fraga
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Chemistry ,Resolution (electron density) ,Trimer ,Protonation ,General Chemistry ,010402 general chemistry ,Ring (chemistry) ,01 natural sciences ,Biochemistry ,Catalysis ,0104 chemical sciences ,Microsecond ,Colloid and Surface Chemistry ,Chemical physics ,Picosecond ,Magic angle spinning ,Spinning - Abstract
Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize the dynamics of phenylalanine residues using 1H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artifact-free dynamics measurements, and allows probing motions effectively without molecular weight limitations. The application to the TET2 enzyme assembly of ∼0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (picoseconds to milliseconds). We quantitatively probe ring-flip motions and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first 13C R1ρ MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as the exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly 13C-labeled samples.
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- 2019
5. Reply on RC3
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Masatsune Kainosho
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- 2021
6. Reply on RC1
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Masatsune Kainosho
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- 2021
7. Reply on RC2
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Masatsune Kainosho
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- 2021
8. Conformational features and ionization states of Lys side chains in a protein revealed by the stereo-array isotope labeling (SAIL) method
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Mitsuhiro Takeda, Yohei Miyanoiri, Tsutomu Terauchi, and Masatsune Kainosho
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TheoryofComputation_ANALYSISOFALGORITHMSANDPROBLEMCOMPLEXITY - Abstract
Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated by NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of Staphylococcal nuclease (SNase), which contains as many as 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of ~5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereo-specifically assigned by using the stereo-array isotope labeled Lys (SAIL-Lys), [U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys. The unambiguously assigned NMR signals for the β-, γ-, δ- and ε-methylene moieties afforded a variety of crucial structural information, which could not be obtained by other methods. For example, the 13Cε signals in the SNase variant, selectively labeled with [ε-13C; ε,ε-D2]-Lys, were ~0.3 ppm up-field shifted in D2O, as compared to those in H2O, except for Lys-66, which showed a ~0.2 ppm up-field shift in D2O. This result indicates that the deuterium-induced up-field shifts of the 13Cε signals depend on the ionization states of the ζ-amino group; i.e., ~ −0.3 ppm for Δδ13Cε [NζD3+-NζH3+] and ~ −0.2 ppm for Δδ13Cε [NζD2-NζH2]. Since the highly sensitive 1D-13C NMR spectrum of a protein selectively labeled with [ε-13C; ε,ε-D2]-Lys shows extremely narrow, well-dispersed 13C signals, the deuterium-induced isotope shifts will be a powerful alternative tool to characterize the ionization states of the Lys ζ-amino groups in larger proteins.
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- 2021
9. Stereo-Array Isotope Labeling (SAIL) and Related Methods
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Masatsune Kainosho
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- 2020
10. Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2
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Werner Kremer, Claudia Elisabeth Munte, Edson Crusca, Markus Beck Erlach, Masatsune Kainosho, Hans Robert Kalbitzer, and Joerg Koehler
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Models, Molecular ,Proton ,Arginine ,Protein Conformation ,1H ,N-15 CHEMICAL-SHIFTS ,GLY-X-ALA ,STEREOSPECIFIC ASSIGNMENT ,H-1-NMR PARAMETERS ,PROTEIN-STRUCTURE ,AQUEOUS-SOLUTIONS ,AMIDE PROTONS ,AMINO-ACIDS ,H-1 ,TETRAPEPTIDES ,High pressure NMR ,Pressure coefficients ,model peptides ,Random-coil ,Chemical shift ,15N ,multi-state equilibria ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,chemistry.chemical_compound ,RESSONÂNCIA MAGNÉTICA NUCLEAR ,Pressure ,Side chain ,570 Biowissenschaften, Biologie ,Amino Acid Sequence ,Amino Acids ,Guanidine ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,chemistry.chemical_classification ,010405 organic chemistry ,Resonance ,Hydrogen Bonding ,Models, Theoretical ,Random coil ,0104 chemical sciences ,Amino acid ,Crystallography ,chemistry ,ddc:570 ,Protons ,Peptides ,Algorithms ,Hydrogen - Abstract
For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of 1H and 15N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficients B1 and B2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1H and 15N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For Hα and HN correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients.
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- 2020
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11. Perspective: next generation isotope-aided methods for protein NMR spectroscopy
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Masatsune Kainosho, Yohei Miyanoiri, Tsutomu Terauchi, and Mitsuhiro Takeda
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0301 basic medicine ,Protein Conformation ,Supramolecular chemistry ,TROSY by isotope labeling ,Isotope-aided NMR method for larger proteins ,010402 general chemistry ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Amino Acids, Aromatic ,Computational chemistry ,Amide ,Amino Acids ,Spectroscopy ,Nuclear Magnetic Resonance, Biomolecular ,Carbon Isotopes ,Isotope ,Chemistry ,Nuclear magnetic resonance spectroscopy ,SAIL aromatic 13CH TROSY ,0104 chemical sciences ,SAIL aliphatic 13CH TROSY ,030104 developmental biology ,Membrane protein ,Structural biology ,Heteronuclear molecule ,Isotope Labeling ,Perspective ,1H-direct observation at ultrahigh-field ,Stereo-array isotope labeling (SAIL) - Abstract
In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of “TROSY by isotope labeling” has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52–57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12C-atoms give profoundly narrow 1H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the 1H-signals are acquired via the spin-coupled 13C- and/or 15N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional 1H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.
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- 2018
12. A Numb–Mdm2 fuzzy complex reveals an isoform-specific involvement of Numb in breast cancer
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Veronica D'Uva, Valentina Cecatiello, Davide Disalvatore, Andrea Basile, Pier Paolo Di Fiore, Salvatore Pece, Michael Sattler, Lee Freiburger, Daniela Tosoni, Maria Grazia Malabarba, Manuela Vecchi, Stefano Confalonieri, Marina Mapelli, Chun Jiun Yang, Ivan Nicola Colaluca, and Masatsune Kainosho
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0301 basic medicine ,Gene isoform ,animal structures ,Notch signaling pathway ,Breast Neoplasms ,Nerve Tissue Proteins ,Article ,03 medical and health sciences ,Exon ,Humans ,Protein Isoforms ,Research Articles ,biology ,Alternative splicing ,fungi ,Membrane Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,ddc ,Ubiquitin ligase ,030104 developmental biology ,RNA splicing ,embryonic structures ,NUMB ,biology.protein ,Cancer research ,Mdm2 ,Tumor Suppressor Protein p53 ,hormones, hormone substitutes, and hormone antagonists - Abstract
Numb regulates the activity of the tumor suppressor p53 by inhibiting Mdm2. This study from Colaluca et al. highlights the structural and molecular bases of Numb–Mdm2 interaction and shows how Numb splicing impacts specifically on p53 regulation and breast cancer prognosis., Numb functions as an oncosuppressor by inhibiting Notch signaling and stabilizing p53. This latter effect depends on the interaction of Numb with Mdm2, the E3 ligase that ubiquitinates p53 and commits it to degradation. In breast cancer (BC), loss of Numb results in a reduction of p53-mediated responses including sensitivity to genotoxic drugs and maintenance of homeostasis in the stem cell compartment. In this study, we show that the Numb–Mdm2 interaction represents a fuzzy complex mediated by a short Numb sequence encompassing its alternatively spliced exon 3 (Ex3), which is necessary and sufficient to inhibit Mdm2 and prevent p53 degradation. Alterations in the Numb splicing pattern are critical in BC as shown by increased chemoresistance of tumors displaying reduced levels of Ex3-containing isoforms, an effect that could be mechanistically linked to diminished p53 levels. A reduced level of Ex3-less Numb isoforms independently predicts poor outcome in BCs harboring wild-type p53. Thus, we have uncovered an important mechanism of chemoresistance and progression in p53-competent BCs.
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- 2018
13. Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific
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Diego F, Gauto, Pavel, Macek, Alessandro, Barducci, Hugo, Fraga, Audrey, Hessel, Tsutomu, Terauchi, David, Gajan, Yohei, Miyanoiri, Jerome, Boisbouvier, Roman, Lichtenecker, Masatsune, Kainosho, and Paul, Schanda
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Carbon Isotopes ,Protein Conformation ,Thermodynamics ,Protons ,Pyrococcus horikoshii ,Aminopeptidases ,Nuclear Magnetic Resonance, Biomolecular ,Article - Abstract
Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize dynamics of phenylalanine residues using (1)H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artefact-free dynamics measurements, and allows probing motions effectively without molecular-weight limitations. The application to the TET2 enzyme assembly of ~0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (ps-ms). We quantitatively probe ring flip motions, and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first (13)C R(1ρ) MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly (13)C-labeled samples.
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- 2019
14. 13C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)
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Mitsuhiro Takeda, Yohei Miyanoiri, Masatsune Kainosho, and Tsutomu Terauchi
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0301 basic medicine ,chemistry.chemical_element ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Chemical reaction ,03 medical and health sciences ,Aprotinin ,medicine ,Animals ,Organic chemistry ,Disulfides ,Carbon-13 Magnetic Resonance Spectroscopy ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Molecular Structure ,Chemistry ,Carbon-13 ,Relaxation (NMR) ,Temperature ,Carbon-13 NMR ,Resonance (chemistry) ,Trypsin ,0104 chemical sciences ,Isoenzymes ,Crystallography ,030104 developmental biology ,Models, Chemical ,Thermodynamics ,Cattle ,Mutant Proteins ,Carbon ,Isomerization ,Algorithms ,medicine.drug - Abstract
Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14–Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D 13C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically 13C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14–Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m c14 and m c38). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for 13C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m c38 isomerization, the 1H-13C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.
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- 2016
15. Recent developments in isotope-aided NMR methods for supramolecular protein complexes –SAIL aromatic TROSY
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Yohei Miyanoiri, Masatsune Kainosho, Mitsuhiro Takeda, and Tsutomu Terauchi
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0301 basic medicine ,Magnetic Resonance Spectroscopy ,Macromolecular Substances ,Stereochemistry ,Phenylalanine ,Biophysics ,Supramolecular chemistry ,010402 general chemistry ,Ring (chemistry) ,01 natural sciences ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,Amide ,Escherichia coli ,Lipid bilayer ,Molecular Biology ,chemistry.chemical_classification ,Carbon Isotopes ,Chemical shift ,Relaxation (NMR) ,Malate Synthase ,Tryptophan ,Proteins ,0104 chemical sciences ,Amino acid ,030104 developmental biology ,chemistry ,Structural biology ,Mutation ,Protons ,Peptides - Abstract
Background The structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. Methods The 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. Results The MSG proteins, selectively labeled with either [δ1,e1,e3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. Conclusions We demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. General significance The method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.
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- 2020
16. Evolution and diversification of the plant gibberellin receptor GID1
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Masatsune Kainosho, Makoto Matsuoka, Sayaka Takehara, Takaaki Hirai, Miyako Ueguchi-Tanaka, Mayuko Kawamura, Takashi Akagi, Eiichi Tanimoto, Hideki Yoshida, Yohei Miyanoiri, Ko Hirano, Mitsuhiro Takeda, and Rie Mitani
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0106 biological sciences ,0301 basic medicine ,diversification ,receptor ,Mutant ,Arabidopsis ,Plant Biology ,Receptors, Cell Surface ,adaptation ,Biology ,01 natural sciences ,Evolution, Molecular ,03 medical and health sciences ,Species Specificity ,Phylogenetics ,Molecular evolution ,evolution ,Phylogeny ,Genetics ,Plant evolution ,chemistry.chemical_classification ,Multidisciplinary ,Phylogenetic tree ,Arabidopsis Proteins ,food and beverages ,Biological Sciences ,biology.organism_classification ,Adaptation, Physiological ,gibberellin ,Amino acid ,030104 developmental biology ,chemistry ,PNAS Plus ,Gibberellin ,010606 plant biology & botany - Abstract
Significance The plant gibberellin receptor GID1 shows sequence similarity to carboxylesterase, suggesting that it is derived from an enzyme. However, how GID1 evolved and was modified is unclear. We identified two amino acids that are essential for GID1 activity, and we found that adjustment of these residues caused GID1 to recognize novel GAs carrying 13-OH as active GAs and to strictly refuse inactive GAs. Phylogenetic analysis of 169 GID1s revealed seven subtypes, and the B-type in core eudicots showed unique characteristics. In fact, certain B-type GID1s showed a higher nonsynonymous-to-synonymous divergence ratio in the region determining GA affinity. Such B-type GID1s with higher affinity were preferentially expressed in the roots in some core eudicot plants and conferred adaptive growth under stress., 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 GA4, 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.
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- 2018
17. [Recent advances in NMR methods for determining the structures and dynamics of larger proteins]
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Youhei, Miyanoiri, Mitsuhiro, Takeda, and Masatsune, Kainosho
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Structure-Activity Relationship ,Protein Conformation ,Drug Design ,Animals ,Proteins ,Nuclear Magnetic Resonance, Biomolecular - Published
- 2018
18. Stable-Isotope-Aided NMR Spectroscopy
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Mitsuhiro Takeda, Masatsune Kainosho, and Yohei Miyanoiri
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0301 basic medicine ,Deuterium NMR ,03 medical and health sciences ,030104 developmental biology ,Stable isotope ratio ,Chemistry ,Analytical chemistry ,Transverse relaxation-optimized spectroscopy ,Fluorine-19 NMR ,Nuclear magnetic resonance spectroscopy ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences - Published
- 2018
19. Isotope-Aided Methods for Biological NMR Spectroscopy: Past, Present, and Future
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Masatsune Kainosho, Yohei Miyanoiri, and Mitsuhiro Takeda
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Hydrogen exchange ,Membrane protein ,Ultrahigh field ,Ring flip ,010405 organic chemistry ,Chemistry ,Nanotechnology ,Nuclear magnetic resonance spectroscopy ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences - Abstract
This chapter starts by providing a historical background of our research endeavors over the past half-century to develop various isotope-aided methods in biological NMR spectroscopy, since innovations bloom only on the rich ground cultivated by previous investigators. We then focused on the stereo-array isotope-labeling (SAIL) method, one of our recent accomplishments, which culminates the isotope-aided NMR technologies for structural studies of proteins from various aspects: accurate structural determinations of large proteins, elaboration for automated structural determination, highly efficient and versatile residue-selective methyl labeling with newly developed auxotrophic E. coli strains, large-amplitude slow-breathing motion (LASBM) as revealed by the aromatic ring flipping of the residues in ligand-binding interfaces, and applications of the deuterium-induced 13C-NMR isotope shift to investigate the hydrogen exchange phenomena of side-chain polar groups. Meanwhile, the expected role of NMR spectroscopy has been rapidly shifting from structure determinations to dynamics studies of biologically interesting targets, such as membrane proteins and larger protein complexes. The dynamic aspects of protein–protein and protein–ligand interactions are closely related to their biological functions and can be efficiently studied by using proteins residue selectively labeled with amino acids bearing optimized labeling patterns, prepared by cellular expression. We are absolutely confident that biological NMR spectroscopy will continually develop with further innovations of isotope-labeling technologies in the coming era, featuring ultrahigh field spectrometers beyond 1 GHz.
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- 2017
20. Nano-mole scale sequential signal assignment by 1H-detected protein solid-state NMR
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Masatsune Kainosho, Yuki Endo, Yiling Xiao, Yoshitaka Ishii, Sudhakar Parthasarathy, Isamu Matsuda, Yusuke Nishiyama, Kazuo Yamauchi, Fei Long, Mitsuhiro Takeda, Tetsuo Asakura, Tsutomu Terauchi, Takahiro Nemoto, and Songlin Wang
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Chemistry ,Proton Magnetic Resonance Spectroscopy ,Metals and Alloys ,Analytical chemistry ,Proteins ,Protonation ,General Chemistry ,Time saving ,Signal ,Article ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Solid-state nuclear magnetic resonance ,Nano ,Mole ,Materials Chemistry ,Ceramics and Composites ,Spinning - Abstract
We present a 3D (1)H-detected solid-state NMR (SSNMR) approach for main-chain signal assignments of 10-100 nmol of fully protonated proteins using ultra-fast magic-angle spinning (MAS) at ∼80 kHz by a novel spectral-editing method, which permits drastic spectral simplification. The approach offers ∼110 fold time saving over a traditional 3D (13)C-detected SSNMR approach.
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- 2015
21. Pressure dependence of side chain
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Markus, Beck Erlach, Joerg, Koehler, Edson, Crusca, Claudia E, Munte, Masatsune, Kainosho, Werner, Kremer, and Hans Robert, Kalbitzer
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Carbon Isotopes ,Magnetic Resonance Spectroscopy ,Models, Chemical ,Pressure ,Amino Acids ,Peptides - Abstract
For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of
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- 2017
22. Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na
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Yohei, Miyanoiri, Atsushi, Hijikata, Yuuki, Nishino, Mizuki, Gohara, Yasuhiro, Onoue, Seiji, Kojima, Chojiro, Kojima, Tsuyoshi, Shirai, Masatsune, Kainosho, and Michio, Homma
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Models, Molecular ,Bacterial Proteins ,Protein Domains ,Flagella ,Sodium ,Point Mutation ,Molecular Dynamics Simulation ,Nuclear Magnetic Resonance, Biomolecular ,Protein Structure, Secondary ,Vibrio alginolyticus - Abstract
The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliG
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- 2017
23. Pressure dependence of side chain 13C chemical shifts in model peptides Ac-Gly-Gly-Xxx-Ala-NH2
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Masatsune Kainosho, Werner Kremer, Hans Robert Kalbitzer, Edson Crusca, Claudia Elisabeth Munte, Joerg Koehler, Markus Beck Erlach, University of Regensburg, Universidade Estadual Paulista (Unesp), Universidade de São Paulo (USP), and Tokyo Metropolitan University
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chemistry.chemical_classification ,Atmospheric pressure ,010405 organic chemistry ,Stereochemistry ,Chemical structure ,Chemical shift ,Stereospecific assignment ,PEPTÍDEOS ,Nuclear magnetic resonance spectroscopy ,Pressure coefficients ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Amino acid ,13C shifts ,Biosynthetically labeled ,chemistry ,High pressure NMR ,Atom ,Side chain ,Random coil peptides ,Conformational isomerism ,Spectroscopy - Abstract
Made available in DSpace on 2018-12-11T16:49:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Deutsche Forschungsgemeinschaft Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of 13C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B1 and B2 is dependent on the type of atom and amino acid studied. For HN, N and Cα the first order pressure coefficient B1 is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically 13C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons. Institute of Biophysics and Physical Biochemistry and Centre of Magnetic Resonance in Chemistry and Biomedicine University of Regensburg Institute of Chemistry São Paulo State University (UNESP) Physics Institute of São Carlos University of São Paulo Graduate School of Science and Technology Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji Institute of Chemistry São Paulo State University (UNESP) Deutsche Forschungsgemeinschaft: FOR1979 Deutsche Forschungsgemeinschaft: Ka 647
- Published
- 2017
24. Expression and purification of a GRAS domain of SLR1, the rice DELLA protein
- Author
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Miyako Ueguchi-Tanaka, Ko Hirano, Youichi Naoe, Hiroaki Kato, Rie Mitani, Makoto Matsuoka, Sayaka Takehara, Tomomi Sato, Yohei Miyanoiri, Mitsuhiro Takeda, and Masatsune Kainosho
- Subjects
Alanine ,medicine.diagnostic_test ,Protein Stability ,Proteolysis ,Oryza ,Biology ,medicine.disease_cause ,Peptide Fragments ,Recombinant Proteins ,Protein Structure, Tertiary ,Biochemistry ,Escherichia coli ,medicine ,Trypsin ,Protein folding ,Overproduction ,Receptor ,Function (biology) ,Plant Proteins ,Biotechnology ,Cysteine - Abstract
GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in Escherichia coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.
- Published
- 2014
25. Molecular Bases of Multimodal Regulation of a Fungal Transient Receptor Potential (TRP) Channel
- Author
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Isamu Yabe, Atsuko Yamashita, Makoto Ihara, Nobuyuki Uozumi, Yohei Miyanoiri, Masatsune Kainosho, Mitsuhiro Takeda, and Shin Hamamoto
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Coiled coil ,Membrane potential ,Fungal protein ,Gibberella ,Chemistry ,Cell Biology ,respiratory system ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Protein Structure, Tertiary ,Fungal Proteins ,TRPC1 ,Structure-Activity Relationship ,Transient receptor potential channel ,TRPC Cation Channels ,Biophysics ,Calcium ,CTD ,human activities ,Molecular Biology ,Ion channel ,Signal Transduction - Abstract
Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca2+ elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module. Background: Multimodality of TRP channels underlies their diverse physiological functions. Results: We identified a fungal multimodal TRP channel whose cytosolic domain (CTD) mediates various channel regulation. Conclusion: CTD has an oligomerization module critical for osmoreception, yet its flexible structure allows dynamic regulations with other functional modalities. Significance: This work proposes structural and biophysical principles for multimodality of a TRP channel family member.
- Published
- 2013
26. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain
- Author
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Masayori Inouye, Yohei Miyanoiri, Tsutomu Terauchi, Yojiro Ishida, Mitsuhiro Takeda, and Masatsune Kainosho
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,Auxotrophy ,Mutant ,Leucines ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,Valine ,Leucine ,Escherichia coli ,Isoleucine ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,chemistry.chemical_classification ,Proteins ,Amino acid ,030104 developmental biology ,chemistry ,Dehydratase ,Isotope Labeling ,Genetic Engineering - Abstract
We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.
- Published
- 2016
27. Differential Large-Amplitude Breathing Motions in the Interface of FKBP12-Drug Complexes
- Author
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Chun Jiun Yang, Masatsune Kainosho, Mitsuhiro Takeda, Tsutomu Terauchi, and Jun-Goo Jee
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Models, Molecular ,Sirolimus ,Ring flip ,Chemistry ,Hydrogen bond ,Ligand ,Protein Conformation ,Hydrogen Bonding ,Plasma protein binding ,Tacrolimus Binding Protein 1A ,Ring (chemistry) ,Ligands ,Biochemistry ,Tacrolimus ,Crystallography ,Motion ,FKBP ,Protein structure ,Amplitude ,Humans ,Thermodynamics ,Immunosuppressive Agents ,Protein Binding - Abstract
The tight complexes FKBP12 forms with immunosuppressive drugs, such as FK506 and rapamycin, are frequently used as models for developing approaches to structure-based drug design. Although the interfaces between FKBP12 and these ligands are well-defined structurally and are almost identical in the X-ray crystallographic structures of various complexes, our nuclear magnetic resonance studies have revealed the existence of substantial large-amplitude motions in the FKBP12-ligand interfaces that depend on the nature of the ligand. We have monitored these motions by measuring the rates of Tyr and Phe aromatic ring flips, and hydroxyl proton exchange for residues clustered within the FKBP12-ligand interface. The results show that the rates of hydroxyl proton exchange and ring flipping for Tyr26 are much slower in the FK506 complex than in the rapamycin complex, whereas the rates of ring flipping for Phe48 and Phe99 are significantly faster in the FK506 complex than in the rapamycin complex. The apparent rate differences observed for the interfacial aromatic residues in the two complexes confirm that these dynamic processes occur without ligand dissociation. We tentatively attribute the differential interface dynamics for these complexes to a single hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen of rapamycin, which is not present in the KF506 complex. This newly identified Phe46 ζ-hydrogen bond in the rapamycin complex imposes motional restriction on the surrounding hydrophobic cluster and subsequently regulates the dynamics within the protein-ligand interface. Such information concerning large-amplitude dynamics at drug-target interfaces has the potential to provide novel clues for drug design.
- Published
- 2015
28. Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins
- Author
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Mitsuhiro Takeda, Tsutomu Terauchi, and Masatsune Kainosho
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Proton ,Protein Conformation ,Biochemistry ,chemistry.chemical_compound ,medicine ,Animals ,Organic chemistry ,Cysteine ,Disulfides ,Methylene ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,chemistry.chemical_classification ,Geminal ,Carbon-13 ,Proteins ,Deuterium ,Trypsin ,Amino acid ,Crystallography ,chemistry ,Spin diffusion ,Cattle ,Protons ,Trypsin Inhibitors ,medicine.drug - Abstract
NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-(13)C, (15)N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β-(13)C; α,β-(2)H(2)] Cys and (2R, 3R)-[β-(13)C; α,β-(2)H(2)] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ(2) and χ(3), can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.
- Published
- 2011
29. Hydrogen Exchange Study on the Hydroxyl Groups of Serine and Threonine Residues in Proteins and Structure Refinement Using NOE Restraints with Polar Side-Chain Groups
- Author
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Masatsune Kainosho, Tsutomu Terauchi, Mitsuhiro Takeda, Akira Ono, and Jun-Goo Jee
- Subjects
Threonine ,Hydrogen exchange ,Protein Conformation ,Stereochemistry ,Chemistry ,Deuterium Exchange Measurement ,Proteins ,General Chemistry ,Isomerase ,Biochemistry ,Catalysis ,Serine ,Colloid and Surface Chemistry ,Hydroxides ,Side chain ,Organic chemistry ,Polar ,Tyrosine ,Nuclear Magnetic Resonance, Biomolecular ,Cysteine - Abstract
We recently developed new NMR methods for monitoring the hydrogen exchange rates of tyrosine hydroxyl (Tyr-OH) and cysteine sulfhydryl (Cys-SH) groups in proteins. These methods facilitate the identification of slowly exchanging polar side-chain protons in proteins, which serve as sources of NOE restraints for protein structure refinement. Here, we have extended the methods for monitoring the hydrogen exchange rates of the OH groups of serine (Ser) and threonine (Thr) residues in an 18.2 kDa protein, EPPIb, and thus demonstrated the usefulness of NOE restraints with slowly exchanging OH protons for refining the protein structure. The slowly exchanging Ser/Thr-OH groups were readily identified by monitoring the (13)C(β)-NMR signals in an H(2)O/D(2)O (1:1) mixture, for the protein containing Ser/Thr residues with (13)C, (2)H-double labels at their β carbons. Under these circumstances, the OH groups exist in equilibrium between the protonated and deuterated isotopomers, and the (13)C(β) peaks of the two species are resolved when their exchange rate is slower than the time scale of the isotope shift effect. In the case of EPPIb dissolved in 50 mM sodium phosphate buffer (pH 7.5) at 40 °C, one Ser and four Thr residues were found to have slowly exchanging hydroxyl groups (k(ex)~40 s(-1)). With the information for the slowly exchanging Ser/Thr-OH groups in hand, we could collect additional NOE restraints for EPPIb, thereby making a unique and important contribution toward defining the spatial positions of the OH protons, and thus the hydrogen-bonding acceptor atoms.
- Published
- 2011
30. Alternative SAIL-Trp for robust aromatic signal assignment and determination of the χ2 conformation by intra-residue NOEs
- Author
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Jun-Goo Jee, Akira M. Ono, Yohei Miyanoiri, Mitsuhiro Takeda, Masatsune Kainosho, Tsutomu Terauchi, and Kosuke Okuma
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Indole test ,Protein Conformation ,Chemistry ,Stereochemistry ,Carbon-13 ,Tryptophan ,Proteins ,Ring (chemistry) ,Resonance (chemistry) ,Biochemistry ,Amino acid ,Proto-Oncogene Proteins c-myb ,Residue (chemistry) ,Protein structure ,Isotope Labeling ,Animals ,Amino Acids ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U-(13)C,(15)N]-proteins, the observation and assignment of the ring signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR assignment, exclusively through the (13)C-(13)C and (13)C-(1)H spin coupling networks (Kainosho et al. in Nature 440:52-57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-(2)H(2); δ1,ε3,η2-(13)C(3); ε1-(15)N]-indole ring ([(12)C (γ,) ( 12) C(ε2)] SAIL-Trp), which provides a more robust way to correlate the (1)H(β), (1)H(α), and (1)H(N) to the (1)H(δ1) and (1)H(ε3) through the intra-residue NOEs. The assignment of the (1)H(δ1)/(13)C(δ1) and (1)H(ε3)/(13)C(ε3) signals can thus be transferred to the (1)H(ε1)/(15)N(ε1) and (1)H(η2)/(13)C(η2) signals, as with the previous type of SAIL-Trp, which has an extra (13)C at the C(γ) of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was (1)H(β2) in this experiment, one can determine the side-chain conformation of the Trp residue including the χ(2) angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [(12)C(γ),(12)C(ε2)] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.
- Published
- 2011
31. Isotope Labelling
- Author
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Mitsuhiro Takeda and Masatsune Kainosho
- Published
- 2011
32. Exclusively NOESY-based automated NMR assignment and structure determination of proteins
- Author
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Yoshiki Shigemitsu, Teppei Ikeya, Masatsune Kainosho, Junpei Hamatsu, Masaki Mishima, Yutaka Ito, Jun-Goo Jee, and Peter Güntert
- Subjects
Models, Molecular ,biology ,Protein Conformation ,Chemistry ,Reproducibility of Results ,Cyana ,biology.organism_classification ,Biochemistry ,Solution structure ,Crystallography ,Protein structure ,Bacterial Proteins ,Fully automated ,Computer Simulation ,Nuclear Magnetic Resonance, Biomolecular ,Two-dimensional nuclear magnetic resonance spectroscopy ,Spectroscopy - Abstract
A fully automated method is presented for determining NMR solution structures of proteins using exclusively NOESY spectra as input, obviating the need to measure any spectra only for obtaining resonance assignments but devoid of structural information. Applied to two small proteins, the approach yielded structures that coincided closely with conventionally determined structures.
- Published
- 2011
33. Detection of the Sulfhydryl Groups in Proteins with Slow Hydrogen Exchange Rates and Determination of Their Proton/Deuteron Fractionation Factors Using the Deuterium-Induced Effects on the 13Cβ NMR Signals
- Author
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Masatsune Kainosho, Jun-Goo Jee, Tsutomu Terauchi, and Mitsuhiro Takeda
- Subjects
Carbon Isotopes ,Proton ,Chemistry ,Stereochemistry ,Chemical shift ,Proteins ,General Chemistry ,Isomerase ,Deuterium ,Biochemistry ,Catalysis ,Isotopomers ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Nuclear magnetic resonance ,Amide ,Cysteine ,Protons ,Nuclear Magnetic Resonance, Biomolecular ,Two-dimensional nuclear magnetic resonance spectroscopy ,Hydrogen - Abstract
A method for identifying cysteine (Cys) residues with sulfhydryl (SH) groups exhibiting slow hydrogen exchange rates has been developed for proteins in aqueous media. The method utilizes the isotope shifts of the C(beta) chemical shifts induced by the deuteration of the SH groups. The 18.2 kDa E. coli peptidyl prolyl cis-trans isomerase b (EPPIb), which was selectively labeled with [3-(13)C;3,3-(2)H(2)]Cys, showed much narrower line widths for the (13)C(beta) NMR signals, as compared to those of the proteins labeled with either [3-(13)C]Cys or (3R)-[3-(13)C;3-(2)H]Cys. The (13)C(beta) signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-(13)C;3,3-(2)H(2)]Cys, split into four signals in H(2)O/D(2)O (1:1) at 40 degrees C and pH 7.5, indicating that the exchange rates of the side-chain SH's and the backbone amides are too slow to average the chemical shift differences of the (13)C(beta) signals, due to the two- and three-bond isotope shifts. By virtue of the well-separated signals, the proton/deuteron fractional factors for both the SH and amide groups of the two Cys residues in EPPIb could be directly determined, as approximately 0.4-0.5 for [SD]/[SH] and 0.9-1.0 for [ND]/[NH], by the relative intensities of the NMR signals for the isotopomers. The proton NOE's of the two slowly exchanging SH's were clearly identified in the NOESY spectra and were useful for the determining the local structure of EPPIb around the Cys residues.
- Published
- 2010
34. 1 H-detected 1 H− 1 H correlation spectroscopy of a stereo-array isotope labeled amino acid under fast magic-angle spinning
- Author
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Hideo Akutsu, Toshimichi Fujiwara, Masatsune Kainosho, and Hiroki Takahashi
- Subjects
Nuclear and High Energy Physics ,Magnetic Resonance Spectroscopy ,Staining and Labeling ,Isotope ,Chemistry ,Biophysics ,Analytical chemistry ,Stereoisomerism ,Valine ,Condensed Matter Physics ,Biochemistry ,Dipole ,Proton NMR ,Magic angle spinning ,Spin Labels ,Protons ,Polarization (electrochemistry) ,Spectroscopy ,Spinning ,Two-dimensional nuclear magnetic resonance spectroscopy ,Algorithms - Abstract
The combined use of selective deuteration, stereo-array isotope labeling (SAIL), and fast magic-angle spinning effectively suppresses the 1 H− 1 H dipolar couplings in organic solids. This method provided the high-field 1 H NMR linewidths comparable to those achieved by combined rotation and multiple-pulse spectroscopy. This technique was applied to two-dimensional 1 H-detected 1 H− 1 H polarization transfer CHH experiments of valine. The signal sensitivity for the 1 H-detected CHH experiments was greater than that for the 13 C-detected 1 H− 1 H polarization transfer experiments by a factor of 2–4. We obtained the 1 H− 1 H distances in SAIL valine by CHH experiments with an accuracy of about 0.2 A by using a theory developed for 1 H− 1 H polarization transfer in 13 C-labeled organic compounds.
- Published
- 2010
35. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination
- Author
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Tsutomu Terauchi, Masatsune Kainosho, Akira M. Ono, and Mitsuhiro Takeda
- Subjects
chemistry.chemical_classification ,Carbon Isotopes ,Protein Conformation ,Stereochemistry ,Escherichia coli Proteins ,Phenylalanine ,Proteins ,Aromaticity ,Isomerase ,Peptidylprolyl Isomerase ,Ring (chemistry) ,Biochemistry ,Amino acid ,Isoenzymes ,chemistry.chemical_compound ,Protein structure ,chemistry ,Isotope Labeling ,Aromatic amino acids ,Tyrosine ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.
- Published
- 2009
36. Protein NMR Study Expanded by the SAIL Method
- Author
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Mitsuhiro Takeda and Masatsune Kainosho
- Subjects
Chemistry - Published
- 2009
37. Stable isotope labeling methods for protein NMR spectroscopy
- Author
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Masatsune Kainosho and Shinya Ohki
- Subjects
Deuterium NMR ,Nuclear and High Energy Physics ,Protein structure ,Nuclear magnetic resonance ,Chemistry ,Protein NMR Spectroscopy ,Physical chemistry ,Stable Isotope Labeling ,Fluorine-19 NMR ,Biochemistry ,Spectroscopy ,Analytical Chemistry - Published
- 2008
38. Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAIL-NMR Method
- Author
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Yuan hsiang Chang, Masatsune Kainosho, Yen Lan Hsu, Chung-ke Chang, Mitsuhiro Takeda, Tai Huang Huang, Teppei Ikeya, and Peter Güntert
- Subjects
Models, Molecular ,NP, nucleocapsid protein ,EMSA, electrophoretic mobility shift assay ,Protein Data Bank (RCSB PDB) ,CT, constant time ,SAIL, stereo-array isotope labeling ,Crystallography, X-Ray ,Severe Acute Respiratory Syndrome ,Oligomer ,UL, uniformly labeled ,SARS nucleocapsid protein ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,nucleocapsid packaging ,Peptide sequence ,SAIL-NMR ,Molecular Structure ,Chemistry ,protein structure determination ,Nucleocapsid Proteins ,NMR spectra database ,Ribonucleoproteins ,Severe acute respiratory syndrome-related coronavirus ,Dimerization ,Heteronuclear single quantum coherence spectroscopy ,SARS-CoV, severe acute respiratory syndrome coronavirus ,Molecular Sequence Data ,HSQC, heteronuclear single-quantum coherence ,Article ,poly-dT, poly-deoxythymine ,PDB, Protein Data Bank ,ssDNA, single-stranded DNA ,Coronavirus Nucleocapsid Proteins ,Humans ,Amino Acid Sequence ,Binding site ,NOE, nuclear Overhauser effect ,Protein Structure, Quaternary ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,Binding Sites ,EDTA, ethylenediaminetetraacetic acid ,Protein Structure, Tertiary ,Crystallography ,CTD, C-terminal domain containing residues 248–365 ,Mutagenesis, Site-Directed ,CTD ,NTD, N-terminal domain containing residues 45–181 ,CSD, chemical shift displacement - Abstract
The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform (13)C and (15)N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the beta-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.
- Published
- 2007
39. Automated structure determination of proteins with the SAIL-FLYA NMR method
- Author
-
Peter Güntert, Teppei Ikeya, Masatsune Kainosho, and Mitsuhiro Takeda
- Subjects
Carbon Isotopes ,Nitrogen Isotopes ,biology ,Protein Conformation ,Stable isotope ratio ,Proteins ,Drug design ,Protein engineering ,Cyana ,biology.organism_classification ,General Biochemistry, Genetics and Molecular Biology ,Protein structure ,Structural biology ,Biophysics ,Sample preparation ,Target protein ,Amino Acids ,Biological system ,Nuclear Magnetic Resonance, Biomolecular ,Algorithms ,Software - Abstract
The labeling of proteins with stable isotopes enhances the NMR method for the determination of 3D protein structures in solution. Stereo-array isotope labeling (SAIL) provides an optimal stereospecific and regiospecific pattern of stable isotopes that yields sharpened lines, spectral simplification without loss of information, and the ability to collect rapidly and evaluate fully automatically the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as those that can be analyzed using conventional methods. Here, we describe a protocol for the preparation of SAIL proteins by cell-free methods, including the preparation of S30 extract and their automated structure analysis using the FLYA algorithm and the program CYANA. Once efficient cell-free expression of the unlabeled or uniformly labeled target protein has been achieved, the NMR sample preparation of a SAIL protein can be accomplished in 3 d. A fully automated FLYA structure calculation can be completed in 1 d on a powerful computer system.
- Published
- 2007
40. Optimal isotope labelling for NMR protein structure determinations
- Author
-
Peter Güntert, Yuki Iwashita, Akira Ono, Masatsune Kainosho, Takuya Torizawa, and Tsutomu Terauchi
- Subjects
Models, Molecular ,Calmodulin ,Crystallography, X-Ray ,Protein structure ,Labelling ,Amino Acids ,Spectroscopy ,Nuclear Magnetic Resonance, Biomolecular ,Multidisciplinary ,biology ,Chemistry ,Stable isotope ratio ,Escherichia coli Proteins ,Proteins ,Resonance ,Cyana ,Deuterium ,biology.organism_classification ,Carbon ,Molecular Weight ,NMR spectra database ,Crystallography ,Isotope Labeling ,Periplasmic Binding Proteins ,biology.protein ,Carrier Proteins ,Hydrogen - Abstract
Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.
- Published
- 2006
41. Evaluation of stereo-array isotope labeling (SAIL) patterns for automated structural analysis of proteins with CYANA
- Author
-
Peter Güntert, Masatsune Kainosho, Tsutomu Terauchi, and Teppei Ikeya
- Subjects
biology ,Isotope ,Protein Conformation ,Stable isotope ratio ,Chemistry ,Combined use ,Analytical chemistry ,Relaxation (iterative method) ,General Chemistry ,Cyana ,biology.organism_classification ,Amidohydrolases ,Protein structure ,Calmodulin ,Isotopes ,Molecular size ,Membrane protein ,Isotope Labeling ,General Materials Science ,Amino Acids ,Biological system ,Mathematical Computing ,Nuclear Magnetic Resonance, Biomolecular ,Software ,Bacterial Outer Membrane Proteins - Abstract
Recently we have developed the stereo-array isotope labeling (SAIL) technique to overcome the conventional molecular size limitation in NMR protein structure determination by employing complete stereo- and regiospecific patterns of stable isotopes. SAIL sharpens signals and simplifies spectra without the loss of requisite structural information, thus making large classes of proteins newly accessible to detailed solution structure determination. The automated structure calculation program CYANA can efficiently analyze SAIL-NOESY spectra and calculate structures without manual analysis. Nevertheless, the original SAIL method might not be capable of determining the structures of proteins larger than 50 kDa or membrane proteins, for which the spectra are characterized by many broadened and overlapped peaks. Here we have carried out simulations of new SAIL patterns optimized for minimal relaxation and overlap, to evaluate the combined use of SAIL and CYANA for solving the structures of larger proteins and membrane proteins. The modified approach reduces the number of peaks to nearly half of that observed with uniform labeling, while still yielding well-defined structures and is expected to enable NMR structure determinations of these challenging systems.
- Published
- 2006
42. Phosphorylation-induced conformational change responsible for the function of a myosin phosphatase inhibitor, CPI-17
- Author
-
Rei Takada, David L. Brautigan, Masumi Eto, Masatsune Kainosho, Shinya Ohki, and Masato Shimizu
- Subjects
Hydrophobic effect ,Molecular switch ,Crystallography ,Conformational change ,animal structures ,Tight binding ,Chemistry ,Myosin ,Biophysics ,Phosphorylation ,General Materials Science ,Nuclear magnetic resonance spectroscopy ,Protein kinase C - Abstract
The structures of CPI-17 (Protein kinase-C dependent protein phosphatase-1 (PP1) inhibitor of 17 kDa) in an inactive and an active form have been determined by multidimensional NMR spectroscopy. Comparison of the two structures revealed how the molecular switch turns on at atomic resolution. Using the NMR structure of CPI-17 in the active form, the binding with catalytic domain of PP1 (PP1c) was simulated and the binding model is proposed in this report. When the phospho-Thr38 docks to the catalytic site of PP1, possible interactions for the tight binding are found; one is electrostatic interaction between a negatively charged cluster on phospho-CPI-17 and an acidic groove of PP1c, and the other is hydrophobic interaction between a hydrophobic surface area of phospho-CPI-17 and a hydrophobic groove of PP1c.
- Published
- 2004
43. A New Stable-Isotope-Aided NMR Method for Structural Determinations of Proteins: The SAIL Method
- Author
-
Tsutomu Terauchi, Takuya Torizawa, and Masatsune Kainosho
- Subjects
ComputingMethodologies_PATTERNRECOGNITION ,Computational chemistry ,Chemistry ,Stable isotope ratio ,Analytical chemistry - Abstract
In the post-genomic era the NMR method for structural determinations of proteins has to be high-throughput, accurate, and applicable to larger proteins. Conventional NMR methods, however, do not meet these requirements simultaneously. The stereo-array-isotope-labeling (SAIL) method, which we are currently developing, is an entirely new NMR technique which satisfies all of these criteria.
- Published
- 2004
44. Distinctive Solution Conformation of Phosphatase Inhibitor CPI-17 Substituted with Aspartate at the Phosphorylation-site Threonine Residue
- Author
-
Masatsune Kainosho, Masato Shimizu, Masumi Eto, David L. Brautigan, Rei Takada, and Shinya Ohki
- Subjects
Models, Molecular ,Threonine ,HNCA experiment ,Protein Conformation ,Swine ,Stereochemistry ,Phosphatase ,Muscle Proteins ,Inhibitory Concentration 50 ,Myosin-Light-Chain Phosphatase ,Structure-Activity Relationship ,Protein structure ,Structural Biology ,Protein Phosphatase 1 ,Escherichia coli ,Phosphoprotein Phosphatases ,Animals ,Enzyme Inhibitors ,Phosphorylation ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Helix bundle ,Aspartic Acid ,Binding Sites ,Chemistry ,Protein phosphatase 1 ,Phosphoproteins ,Solutions ,Amino Acid Substitution ,Vascular smooth muscle contraction ,Heteronuclear single quantum coherence spectroscopy - Abstract
We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H–15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.
- Published
- 2003
45. [Untitled]
- Author
-
Sakurako Shimotakahara, Seiji Okubo, Shigeyuki Yokoyama, Heisaburo Shindo, Masatsune Kainosho, Mitsuru Tashiro, Hideyo Yasuda, and Hideki Hatanaka
- Subjects
Genetics ,Parkinson's disease ,Ubiquitin ,biology ,Chemistry ,Product (mathematics) ,medicine ,biology.protein ,Parkin gene ,medicine.disease ,Biochemistry ,Spectroscopy ,Parkin - Published
- 2003
46. [Untitled]
- Author
-
Akira Ono, Jérôme Boisbouvier, Masatsune Kainosho, Ad Bax, and Zhengrong Wu
- Subjects
Chemistry ,Relaxation (NMR) ,Analytical chemistry ,Rotational diffusion ,Thermodynamics ,Nuclear magnetic resonance spectroscopy ,Crystal structure ,Carbon-13 NMR ,Anisotropy ,Biochemistry ,Spectroscopy ,Rotational correlation time ,Diffusion Anisotropy - Abstract
Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)2 from 13C R1ρ and R1 measurements on the C1′, C3′, and C4′ carbons in samples uniformly enriched in 13C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R1ρ/R1 ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R1ρ/R1 ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D||/D⊥, of 2.1±0.4, and an overall rotational correlation time, (2D||+4D⊥)−1, of 3.35 ns at 35 °C in D2O, in excellent agreement with values obtained from hydrodynamic modeling.
- Published
- 2003
47. Automated resonance assignment of the 21kDa stereo-array isotope labeled thioldisulfide oxidoreductase DsbA
- Author
-
Teppei Ikeya, Elena Schmidt, Masatsune Kainosho, Peter Güntert, Yutaka Ito, Mitsuhiro Takeda, Frank Löhr, and Lena Buchner
- Subjects
chemistry.chemical_classification ,Nuclear and High Energy Physics ,biology ,Isotope ,Chemistry ,Stereochemistry ,Chemical shift ,Biophysics ,Analytical chemistry ,Resonance ,Cyana ,Condensed Matter Physics ,biology.organism_classification ,Biochemistry ,DsbA ,Oxidoreductase ,Peak picking ,biology.protein ,Two-dimensional nuclear magnetic resonance spectroscopy - Abstract
The automated chemical shift assignment algorithm FLYA has been extended for use with stereo-array isotope labeled (SAIL) proteins to determine the sequence-specific resonance assignments of large proteins. Here we present the assignment of the backbone and sidechain chemical shifts of the 21 kDa thioldisulfide oxidoreductase DsbA from Escherichia coli that were determined with the SAIL-FLYA algorithm in conjunction with automated peak picking. No manual corrections of peak lists or assignments were applied. The assignments agreed with manually determined reference assignments in 95.4% of the cases if 16 input spectra were used, 94.1% if only 3D 13 C/ 15 N-resolved NOESY, CBCA(CO)NH, and 2D [ 13 C/ 15 N, 1 H]-HSQC were used, and 86.8% if exclusively 3D 13 C/ 15 N-resolved NOESY spectra were used. Considering only the assignments that are classified as reliable by the SAIL-FLYA algorithm, the degrees of agreement increased to 97.5%, 96.5%, and 94.2%, respectively. With our approach it is thus possible to automatically obtain almost complete and correct assignments of proteins larger than 20 kDa.
- Published
- 2014
48. Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation 1 1Edited by P. E. Wright
- Author
-
Yuichiro Hayashi, Eri Kariya, David L. Brautigan, Michio Yazawa, Masumi Eto, Masatsune Kainosho, Shinya Ohki, and Toshiya Hayano
- Subjects
Conformational change ,Myosin light-chain kinase ,Structural Biology ,Stereochemistry ,Chemistry ,Myosin phosphatase activity ,Phosphatase ,Helix ,Myosin ,Phosphorylation ,Molecular Biology ,Heteronuclear single quantum coherence spectroscopy - Abstract
Contractility of vascular smooth muscle depends on phosphorylation of myosin light chains, and is modulated by hormonal control of myosin phosphatase activity. Signaling pathways activate kinases such as PKC or Rho-dependent kinases that phosphorylate the myosin phosphatase inhibitor protein called CPI-17. Phosphorylation of CPI-17 at Thr38 enhances its inhibitory potency 1000-fold, creating a molecular on/off switch for regulating contraction. We report the solution NMR structure of the CPI-17 inhibitory domain (residues 35–120), which retains the signature biological properties of the full-length protein. The final ensemble of 20 sets of NMR coordinates overlaid onto their mean structure with r.m.s.d. values of 0.84(±0.22) A for the backbone atoms. The protein forms a novel four-helix, V-shaped bundle comprised of a central anti-parallel helix pair (B/C helices) flanked by two large spiral loops formed by the N and C termini that are held together by another anti-parallel helix pair (A/D helices) stabilized by intercalated aromatic and aliphatic side-chains. Chemical shift perturbations indicated that phosphorylation of Thr38 induces a conformational change involving displacement of helix A, without significant movement of the other three helices. This conformational change seems to flex one arm of the molecule, thereby exposing new surfaces of the helix A and the nearby phosphorylation loop to form specific interactions with the catalytic site of the phosphatase. This phosphorylation-dependent conformational change offers new structural insights toward understanding the specificity of CPI-17 for myosin phosphatase and its function as a molecular switch.
- Published
- 2001
49. Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca 2+ /calmodulin-dependent kinase kinase peptide 1 1Edited by K. Morikawa
- Author
-
Masatsune Kainosho, Hirofumi Kurokawa, Masanori Osawa, Hiroyuki Kurihara, Mitsuhiko Ikura, Mark B. Swindells, Hiroshi Tokumitsu, and Naoko Katayama
- Subjects
chemistry.chemical_classification ,animal structures ,Myosin light-chain kinase ,Calmodulin ,biology ,EF hand ,Peptide ,Protein structure ,Biochemistry ,chemistry ,Structural Biology ,Ca2+/calmodulin-dependent protein kinase ,Calcium-binding protein ,Biophysics ,biology.protein ,Molecular Biology ,Peptide sequence - Abstract
Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.
- Published
- 2001
50. Synthesis of 13C/D Doubly Labeled <scp>l</scp>-Leucines: Probes for Conformational Analysis of the Leucine Side-chain
- Author
-
Fumiyo Oikawa, Masahito Kobayashi, Makoto Oba, Kozaburo Nishiyama, and Masatsune Kainosho
- Subjects
Carbon Isotopes ,Magnetic Resonance Spectroscopy ,Chemistry ,Stereochemistry ,Organic Chemistry ,Molecular Conformation ,Leucines ,Deuterium ,Molecular conformation ,Leucine ,Isotopes of carbon ,Isotope Labeling ,Side chain - Published
- 2001
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