104 results on '"Kenji Sugase"'
Search Results
2. Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance
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Yasuaki Kabe, Takanori Nakane, Ikko Koike, Tatsuya Yamamoto, Yuki Sugiura, Erisa Harada, Kenji Sugase, Tatsuro Shimamura, Mitsuyo Ohmura, Kazumi Muraoka, Ayumi Yamamoto, Takeshi Uchida, So Iwata, Yuki Yamaguchi, Elena Krayukhina, Masanori Noda, Hiroshi Handa, Koichiro Ishimori, Susumu Uchiyama, Takuya Kobayashi, and Makoto Suematsu
- Subjects
Science - Abstract
PGRMC1 binds to EGFR and cytochromes P450, and is known to be involved in cancer proliferation and in drug resistance. Here, the authors determine the structure of the cytosolic domain of PGRMC1, which forms a dimer via haem–haem stacking, and propose how this interaction could be involved in its function.
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- 2016
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3. Exploration of the Conformational Dynamics of Major Histocompatibility Complex Molecules
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Saeko Yanaka and Kenji Sugase
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major histocompatibility complex ,stability ,nuclear magnetic resonance ,relaxation dispersion ,conformational dynamics ,transient induced-fit model ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Major histocompatibility complex (MHC) molecules are loaded with a wide variety of self- and non-self-peptides in their binding grooves and present these to T cell receptors (TCRs) in order to activate the adaptive immune system. A large number of crystal structures of different MHC alleles with different bound peptides have been determined, and they have been found to be quite similar to one another regardless of the bound peptide sequence. The structures do not change markedly even when forming complexes with TCRs. Nonetheless, the degree of TCR activation does differ markedly depending on the peptide presented by the MHC. Recent structural studies in solution rather than as crystals have suggested that the conformational dynamics of MHC molecules may be responsible for the MHC stability differences. Furthermore, it was shown that the conformational dynamics of MHC molecules is important for peptide loading and presentation to TCR. Here, we describe the static and dynamic structures of MHC molecules and appropriate methods to analyze them. We focus particularly on nuclear magnetic resonance (NMR), one of the most powerful tools to study dynamic properties of proteins. The number of such studies in the literature is limited, but in this review, we show that NMR is valuable for elucidating the structural dynamics of MHC molecules.
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- 2017
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4. Hydrogen-Deuterium Exchange Profiles of Polyubiquitin Fibrils
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Daichi Morimoto, Ryo Nishizawa, Erik Walinda, Shingo Takashima, Kenji Sugase, and Masahiro Shirakawa
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ubiquitin ,amyloid fibrils ,hydrogen-deuterium exchange ,Organic chemistry ,QD241-441 - Abstract
Ubiquitin and its polymeric forms are conjugated to intracellular proteins to regulate diverse intracellular processes. Intriguingly, polyubiquitin has also been identified as a component of pathological protein aggregates associated with Alzheimer’s disease and other neurodegenerative disorders. We recently found that polyubiquitin can form amyloid-like fibrils, and that these fibrillar aggregates can be degraded by macroautophagy. Although the structural properties appear to function in recognition of the fibrils, no structural information on polyubiquitin fibrils has been reported so far. Here, we identify the core of M1-linked diubiquitin fibrils from hydrogen-deuterium exchange experiments using solution nuclear magnetic resonance (NMR) spectroscopy. Intriguingly, intrinsically flexible regions became highly solvent-protected in the fibril structure. These results indicate that polyubiquitin fibrils are formed by inter-molecular interactions between relatively flexible structural components, including the loops and edges of secondary structure elements.
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- 2018
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5. Real-Time Observation of the Interaction between Thioflavin T and an Amyloid Protein by Using High-Sensitivity Rheo-NMR
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Naoto Iwakawa, Daichi Morimoto, Erik Walinda, Yasushi Kawata, Masahiro Shirakawa, and Kenji Sugase
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amyloid fibrils ,thioflavin T ,molecular interactions ,Rheo-NMR ,real-time observation ,SOD1 ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Amyloid fibril formation is associated with numerous neurodegenerative diseases. To elucidate the mechanism of fibril formation, the thioflavin T (ThT) fluorescence assay is widely used. ThT is a fluorescent dye that selectively binds to amyloid fibrils and exhibits fluorescence enhancement, which enables quantitative analysis of the fibril formation process. However, the detailed binding mechanism has remained unclear. Here we acquire real-time profiles of fibril formation of superoxide dismutase 1 (SOD1) using high-sensitivity Rheo-NMR spectroscopy and detect weak and strong interactions between ThT and SOD1 fibrils in a time-dependent manner. Real-time information on the interaction between ThT and fibrils will contribute to the understanding of the binding mechanism of ThT to fibrils. In addition, our method provides an alternative way to analyze fibril formation.
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- 2017
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6. Biological and Physicochemical Functions of Ubiquitylation Revealed by Synthetic Chemistry Approaches
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Daichi Morimoto, Erik Walinda, Kenji Sugase, and Masahiro Shirakawa
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ubiquitin ,post-translational modification ,chemical ubiquitylation ,site-directed conjugation ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Most intracellular proteins are subjected to post-translational modification by ubiquitin. Accordingly, it is of fundamental importance to investigate the biological and physicochemical effects of ubiquitylation on substrate proteins. However, preparation of ubiquitylated proteins by an enzymatic synthesis bears limitations in terms of yield and site-specificity. Recently established chemical ubiquitylation methodologies can overcome these problems and provide a new understanding of ubiquitylation. Herein we describe the recent chemical ubiquitylation procedures with a focus on the effects of ubiquitylation on target proteins revealed by the synthetic approach.
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- 2017
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7. Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity.
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Erik Walinda, Kenji Sugase, Naoki Ishii, Masahiro Shirakawa, Kazuhiro Iwai, and Daichi Morimoto
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UBIQUITIN , *ZINC-finger proteins , *POST-translational modification , *IMMUNOSUPPRESSION , *IMMUNOLOGIC diseases , *CELL death - Abstract
Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain–interacting protein), specifically synthesizes “head-to-tail” poly-Ub chains, which are linked via the N-terminal methionine αamino and C-terminal carboxylate of adjacent Ub units and are thus commonly called “linear” poly-Ub chains. Linear ubiquitin chain assembly complex–assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ub via its Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub–bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain’s role in full-length HOIL-1L. [ABSTRACT FROM AUTHOR]
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- 2023
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8. Rheo‐NMR Spectroscopy for Cryogenic‐Probe‐Equipped NMR Instruments to Monitor Protein Aggregation
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Daichi, Morimoto, Erik, Walinda, Akihiko, Yamamoto, Ulrich, Scheler, and Kenji, Sugase
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Protein Aggregates ,Medical Laboratory Technology ,Magnetic Resonance Spectroscopy ,General Immunology and Microbiology ,Viscosity ,General Neuroscience ,Amyloidogenic Proteins ,Health Informatics ,General Pharmacology, Toxicology and Pharmaceutics ,Magnetic Resonance Imaging ,General Biochemistry, Genetics and Molecular Biology - Abstract
Cryogenic-probe-based Rheo-NMR spectroscopy is a recently developed methodology to obtain solution NMR spectra of protein samples in situ under external shear. It is applicable to atomic-resolution monitoring of protein aggregation in situ, thereby aiding understanding of the transient structural changes and state conversion of amyloidogenic proteins, which are strongly associated with the both the onset and the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Here, we present detailed experimental procedures for the instrumental setup and practical tips for preparation of NMR measurement to analyze protein aggregation by this technique. This protocol will thus aid future Rheo-NMR spectroscopic studies not only of protein aggregation but also of other phenomena related to shear stress, such as shear-induced viscosity increase and shear-enhanced crystallization. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Setup of a Rheo-NMR Instrument Basic Protocol 2: Adjustment of the Vertical and Horizontal Positions of the Glass Stick Basic Protocol 3: Monitoring Protein Aggregation by Rheo-NMR Spectroscopy.
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- 2022
9. Efficient identification and analysis of chemical exchange in biomolecules by R1ρ relaxation dispersion with Amaterasu.
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Erik Walinda, Daichi Morimoto, Mayu Nishizawa, Masahiro Shirakawa, and Kenji Sugase
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- 2016
- Full Text
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10. Corrigendum to 'Molecular recognition and deubiquitination of cyclic K48-linked ubiquitin chains by OTUB1' [Biochem. Biophys. Res. Commun. 562 (2021) 94–99]
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Tomoki Sorada, Daichi Morimoto, Erik Walinda, and Kenji Sugase
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Biophysics ,Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2023
11. Backbone resonance assignments of the A2 domain of mouse von Willebrand factor
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Daichi Morimoto, Erik Walinda, Yutaka Mahana, Masanori Osugi, Masahiro Shirakawa, and Kenji Sugase
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congenital, hereditary, and neonatal diseases and abnormalities ,biology ,Chemistry ,Cleavage (embryo) ,medicine.disease ,Biochemistry ,ADAMTS13 ,Mice ,Von Willebrand factor ,Structural Biology ,hemic and lymphatic diseases ,Hemostasis ,von Willebrand Factor ,cardiovascular system ,biology.protein ,Biophysics ,Von Willebrand disease ,medicine ,Animals ,Peptide bond ,Platelet ,Protein secondary structure ,circulatory and respiratory physiology - Abstract
von Willebrand factor (vWF) is an adhesive plasma protein that is important for platelet adhesion in normal hemostasis in response to vascular injury. Although large vWF multimers are released from storage granules of platelets and (sub-)endothelial cells in response to hemostatic stimuli, for normal physiological function, vWF multimers are required to be cleaved into smaller multimeric forms. The plasma metalloproteinase ADAMTS13 specifically cleaves the peptide bond located in the middle of the A2 domain of vWF (vWF-A2), but the cleavage site is buried inside the structure of vWF and is difficult to access in the absence of elevated flow shear stress. On the other hand, in the presence of high vascular shear stress, the structure of vWF-A2 is supposed to be unfolded, thereby becoming accessible for proteolysis by ADAMTS13. However, the atomic-level mechanism underlying shear-induced structural changes of vWF-A2 remains unclear and to date no solution NMR information is available. In this study, we present the backbone 1H, 13C, and 15N resonance assignments of mouse vWF-A2; side chain assignments of 13Cβ are also provided. Secondary structure propensity analysis based on the assigned chemical shifts showed that mouse vWF-A2 forms similar secondary structures in solution to the previously determined crystal structure of human vWF-A2. The obtained NMR assignment data will contribute to an atomic-level characterization of shear-induced unfolding of vWF-A2 in solution.
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- 2021
12. Transient Diffusive Interactions with a Protein Crowder Affect Aggregation Processes of Superoxide Dismutase 1 β-Barrel
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Kenji Sugase, Masahiro Shirakawa, Daichi Morimoto, Erik Walinda, Sarah Leeb, Jens Danielsson, and Naoto Iwakawa
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animal diseases ,SOD1 ,010402 general chemistry ,01 natural sciences ,Diffusion ,Superoxide dismutase ,Superoxide Dismutase-1 ,0103 physical sciences ,Materials Chemistry ,medicine ,Humans ,Physical and Theoretical Chemistry ,Amyotrophic lateral sclerosis ,010304 chemical physics ,biology ,Superoxide Dismutase ,Chemistry ,Amyotrophic Lateral Sclerosis ,nutritional and metabolic diseases ,A protein ,medicine.disease ,nervous system diseases ,0104 chemical sciences ,Surfaces, Coatings and Films ,Barrel ,nervous system ,Mutation ,biology.protein ,Biophysics ,Muramidase ,Chemical stability - Abstract
Aggregate formation of superoxide dismutase 1 (SOD1) inside motor neurons is known as a major factor in onset of amyotrophic lateral sclerosis. The thermodynamic stability of the SOD1 β-barrel has been shown to decrease in crowded environments such as inside a cell, but it remains unclear how the thermodynamics of crowding-induced protein destabilization relate to SOD1 aggregation. Here we have examined the effects of a protein crowder, lysozyme, on fibril aggregate formation of the SOD1 β-barrel. We found that aggregate formation of SOD1 is decelerated even in mildly crowded solutions. Intriguingly, transient diffusive interactions with lysozyme do not significantly affect the static structure of the SOD1 β-barrel but stabilize an alternative excited "invisible" state. The net effect of crowding is to favor species off the aggregation pathway, thereby explaining the decelerated aggregation in the crowded environment. Our observations suggest that the intracellular environment may have a similar negative (inhibitory) effect on fibril formation of other amyloidogenic proteins in living cells. Deciphering how crowded intracellular environments affect aggregation and fibril formation of such disease-associated proteins will probably become central in understanding the exact role of aggregation in the etiology of these enigmatic diseases.
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- 2021
13. Structural Dynamic Heterogeneity of Polyubiquitin Subunits Affects Phosphorylation Susceptibility
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Shingo Takashima, Daichi Morimoto, Erik Walinda, Kazuhiro Iwai, Kenji Sugase, Masahiro Shirakawa, and Mayu Nishizawa
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Models, Molecular ,0303 health sciences ,biology ,Protein Conformation ,Chemistry ,Kinase ,030302 biochemistry & molecular biology ,Protein tag ,Plasma protein binding ,Biochemistry ,Protein tertiary structure ,03 medical and health sciences ,Protein structure ,Ubiquitin ,biology.protein ,Biophysics ,Humans ,Moiety ,Phosphorylation ,Polyubiquitin ,Protein Kinases ,Protein Processing, Post-Translational ,Protein Binding - Abstract
Polyubiquitin is a multifunctional protein tag formed by the covalent conjugation of ubiquitin molecules. Due to the high rigidity of the ubiquitin fold, the ubiquitin moieties in a polyubiquitin chain appear to be structurally equivalent to each other. It is therefore unclear how a specific ubiquitin moiety in a chain may be preferentially recognized by some proteins, such as the kinase PINK1. Here we show that there is structural dynamic heterogeneity in the two ubiquitin moieties of K48-linked diubiquitin by NMR spectroscopic analyses. Our analyses capture subunit-asymmetric structural fluctuations that are not directly related to the closed-to-open transition of the two ubiquitin moieties in diubiquitin. Strikingly, these newly identified heterogeneous structural fluctuations may be linked to an increase in susceptibility to phosphorylation by PINK1. Coupled with the fact that there are almost no differences in static tertiary structure among ubiquitin moieties in a chain, the observed subunit-specific structural fluctuations may be an important factor that distinguishes individual ubiquitin moieties in a chain, thereby aiding both efficiency and specificity in post-translational modifications.
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- 2021
14. Rigorous analysis of the interaction between proteins and low water-solubility drugs by qNMR-aided NMR titration experiments
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Kenji Sugase, Erik Walinda, Daichi Morimoto, and Takuya Hirakawa
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Drug ,Magnetic Resonance Spectroscopy ,Aqueous solution ,Chemistry ,media_common.quotation_subject ,Water ,General Physics and Astronomy ,Serum Albumin, Bovine ,Tacrolimus Binding Protein 1A ,Nuclear magnetic resonance spectroscopy ,Tacrolimus ,Dissociation constant ,Solubility ,Computational chemistry ,Nmr titration ,Animals ,Cattle ,Target protein ,Physical and Theoretical Chemistry ,Quantitative analysis (chemistry) ,media_common - Abstract
Drugs are designed and validated based on physicochemical data on their interactions with target proteins. For low water-solubility drugs, however, quantitative analysis is practically impossible without accurate estimation of precipitation. Here we combined quantitative NMR with NMR titration experiments to rigorously quantify the interaction of the low water-solubility drug pimecrolimus with its target protein FKBP12. Notably, the dissociation constants estimated with and without consideration of precipitation differed by more than tenfold. Moreover, the method enabled us to quantitate the FKBP12-pimecrolimus interaction even under a crowded condition established using the protein crowder BSA. Notably, the FKBP12-pimecrolimus interaction was slightly hampered under the crowded environment, which is explained by transient association of BSA with the drug molecules. Collectively, the described method will contribute to both quantifying the binding properties of low water-solubility drugs and to elucidating the drug behavior in complex crowded solutions including living cells.
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- 2021
15. Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by 18O-incorporation
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Daichi Morimoto, Yuka Tanaka, Erik Walinda, Kenji Sugase, and Masahiro Shirakawa
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0301 basic medicine ,biology ,Chemistry ,Kinetic analysis ,Biophysics ,Cell Biology ,Conjugated system ,Biochemistry ,Deubiquitinating enzyme ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Ubiquitin ,030220 oncology & carcinogenesis ,biology.protein ,Molecule ,Reactivity (chemistry) ,Molecular Biology ,Intracellular ,Deubiquitination - Abstract
Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied 18O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two 16O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with 18O atoms by deubiquitination in 18O-labeled aqueous solution. By using quantitative mass spectrometry, we detected 18O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the 18O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated 18O-incorporation into ubiquitin.
- Published
- 2020
16. Pinpoint analysis of a protein in slow exchange using F1F2-selective ZZ-exchange spectroscopy: assignment and kinetic analysis
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Daichi Morimoto, Erik Walinda, Kenji Sugase, and Mayu Nishizawa
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0301 basic medicine ,chemistry.chemical_classification ,Chemistry ,Biomolecule ,Protein dynamics ,Kinetic analysis ,A protein ,Nuclear magnetic resonance spectroscopy ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,03 medical and health sciences ,030104 developmental biology ,Chemical physics ,Slow exchange ,Spectroscopy ,Two-dimensional nuclear magnetic resonance spectroscopy - Abstract
ZZ-exchange spectroscopy is widely used to study slow exchange processes in biomolecules, especially determination of exchange rates and assignment of minor peaks. However, if the exchange cross peaks overlap or the populations are skewed, kinetic analysis is hindered. In order to analyze slow exchange protein dynamics under such conditions, here we have developed a new method by combining ZZ-exchange and F1F2-selective NMR spectroscopy. We demonstrate the utility of this method by examining the monomer-dimer transition of the ubiquitin-associated domain of p62, successfully assigning the minor (monomeric) peaks and obtaining the exchange rates, which cannot be achieved by ZZ-exchange alone.
- Published
- 2020
17. Structural Insights into Methylated DNA Recognition by the Methyl-CpG Binding Domain of MBD6 from
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Yutaka, Mahana, Izuru, Ohki, Erik, Walinda, Daichi, Morimoto, Kenji, Sugase, and Masahiro, Shirakawa
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Article - Abstract
Cytosine methylation is an epigenetic modification essential for formation of mature heterochromatin, gene silencing, and genomic stability. In plants, methylation occurs not only at cytosine bases in CpG but also in CpHpG and CpHpH contexts, where H denotes A, T, or C. Methyl-CpG binding domain (MBD) proteins, which recognize symmetrical methyl-CpG dinucleotides and act as gene repressors in mammalian cells, are also present in plant cells, although their structural and functional properties still remain poorly understood. To fill this gap, in this study, we determined the solution structure of the MBD domain of the MBD6 protein from Arabidopsis thaliana and investigated its binding properties to methylated DNA by binding assays and an in-depth NMR spectroscopic analysis. The AtMBD6 MBD domain folds into a canonical MBD structure in line with its binding specificity toward methyl-CpG and possesses a DNA binding interface similar to mammalian MBD domains. Intriguingly, however, the binding affinity of the AtMBD6 MBD domain toward methyl-CpG-containing DNA was found to be much lower than that of known mammalian MBD domains. The main difference arises from the absence of positively charged residues in AtMBD6 that supposedly interact with the DNA backbone as seen in mammalian MBD/methyl-CpG-containing DNA complexes. Taken together, we have established a structural basis for methyl-CpG recognition by AtMBD6 to develop a deeper understanding how MBD proteins work as mediators of epigenetic signals in plant cells.
- Published
- 2021
18. Effects of Weak Nonspecific Interactions with ATP on Proteins
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Masahiro Shirakawa, Erik Walinda, Mayu Nishizawa, Kenji Sugase, Daichi Morimoto, Benjamin Kohn, and Ulrich Scheler
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chemistry.chemical_classification ,Binding Sites ,Magnetic Resonance Spectroscopy ,biology ,Ubiquitin ,Metabolite ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Molecular Dynamics Simulation ,Biochemistry ,Catalysis ,Divalent ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Monomer ,Adenosine Triphosphate ,chemistry ,Sequestosome-1 Protein ,biology.protein ,Biophysics ,alpha-Synuclein ,Non-covalent interactions ,Chelation ,Adenosine triphosphate - Abstract
Adenosine triphosphate (ATP) is an immensely well-studied metabolite serving multiple key biochemical roles as the major chemical energy currency in living systems, a building block of ribonucleic acids, and a phosphoryl group donor in kinase-mediated signaling. Intriguingly, ATP has been recently proposed to act as a hydrotrope that inhibits aggregation of amyloidogenic proteins; however, the underlying mechanism and the general physicochemical effect that coexistence with ATP exerts on proteins remain unclear. By combining NMR spectroscopy and MD simulations, here we observed weak but unambiguously measurable and concentration-dependent noncovalent interactions between ATP and various proteins. The interactions were most pronounced for an intrinsically disordered protein (α-synuclein) and for residues in flexible regions (e.g., loops or termini) of two representative folded proteins (ubiquitin and the dimeric ubiquitin-binding domain of p62). As shown by solution NMR, a consequence of the ATP-protein interaction was altered hydration of solvent-exposed residues in the protein. The observation that ATP interacted with all three proteins suggests that ATP is a general nonspecific binder of proteins. Several complementary biophysical methods further confirmed that, at physiological concentrations of ∼5-10 mM, ATP starts to form oligomeric states via magnesium-chelating and chelation-independent mechanisms, in agreement with previous studies. Although the observed ATP-protein interaction was relatively weak overall, the high ratio of ATP (monomeric free ATP, mono- and divalent ion-bound ATP, oligomeric and chelated ATP) to proteins in cells suggests that most proteins are likely to encounter transient interactions with ATP (and chemically similar metabolites) that confer metabolite-mediated protein surface protection.
- Published
- 2021
19. Multiple-State Monitoring of SOD1 Amyloid Formation at Single-Residue Resolution by Rheo-NMR Spectroscopy
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Daichi Morimoto, Masahiro Shirakawa, Erik Walinda, Kenji Sugase, and Naoto Iwakawa
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Amyloid ,Protein dynamics ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Protein aggregation ,Fibril ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Residue (chemistry) ,Colloid and Surface Chemistry ,Monomer ,chemistry ,Biophysics ,Spectroscopy - Abstract
Formation of protein aggregates or fibrils entails the conversion of soluble native protein monomers via multiple molecular states. No spectroscopic techniques have succeeded in capturing the transient molecular-scale events of fibrillation in situ. Here we report residue- and state-specific real-time monitoring of the fibrillation of amyotrophic lateral sclerosis-related SOD1 by rheology NMR (Rheo-NMR) spectroscopy. Under moderately denaturing conditions, where NMR signals of folded and unfolded monomeric SOD1 are simultaneously observable, the cross-peak intensities of folded monomeric SOD1 decreased faster than those of the unfolded species, and a 310-helix in folded SOD1 was deformed prior to global unfolding. Furthermore, real-time protein dynamics analysis identified residues involved in the core structure formation of SOD1 oligomers. Our findings provide insight into local and global unfolding events in SOD1 and fibril formation. This Rheo-NMR analysis will be applicable not only to atomic-level monitoring of other amyloidogenic proteins but also to quantification of shear-induced structural changes of non-amyloidogenic proteins and elucidation of shear-enhanced chemical phenomena such as viscosity increase and crystallization of various solution-state compounds.
- Published
- 2021
20. Molecular recognition and deubiquitination of cyclic K48-linked ubiquitin chains by OTUB1
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Daichi Morimoto, Tomoki Sorada, Kenji Sugase, and Erik Walinda
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0301 basic medicine ,Ubiquitin binding ,Protein Conformation ,Proton Magnetic Resonance Spectroscopy ,Biophysics ,Cleavage (embryo) ,Biochemistry ,Deubiquitinating enzyme ,03 medical and health sciences ,0302 clinical medicine ,Molecular recognition ,Ubiquitin ,Humans ,Molecular Biology ,chemistry.chemical_classification ,biology ,Deubiquitinating Enzymes ,Nitrogen Isotopes ,Lysine ,Ubiquitination ,Cell Biology ,Kinetics ,030104 developmental biology ,Enzyme ,chemistry ,OTUB1 ,Cyclization ,030220 oncology & carcinogenesis ,biology.protein ,Deubiquitination - Abstract
Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes not only canonical (noncyclic) but also cyclic K48-linked ubiquitin chains. Although the cyclic conformation is expected to repress molecular recognition by ubiquitin binding proteins due to restricting the flexibility of the ubiquitin subunits in a chain, multiple proteins are reported to associate with cyclic ubiquitin chains similar to noncyclic chains. However, the molecular mechanism of how cyclic ubiquitin chains are recognized remains unclear. Here we investigated the effect of cyclization on ubiquitin-chain cleavage and molecular recognition by a K48-linkage specific deubiquitinating enzyme OTUB1 for cyclic diubiquitin by NMR spectroscopic analyses. Compared to noncyclic diubiquitin, we observed slow but unambiguously detectable cleavage of cyclic diubiquitin to monoubiquitin by OTUB1. Intriguingly, upon ubiquitin chain cleavage, cyclic diubiquitin appeared to alter its "autoinhibited" conformation to an incompletely but partially accessible conformation, induced by interaction with OTUB1 via the ubiquitin-subunit specific recognition patches and adjacent surfaces. These data imply that cyclic ubiquitin chains may exist stably in cells in spite of the presence of deubiquitinating enzymes and that these chains can be recognized by intracellular proteins in a manner distinct from that of noncyclic ubiquitin chains.
- Published
- 2021
21. Counter-flow phenomena studied by nuclear magnetic resonance (NMR) velocimetry and flow simulations
- Author
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Benjamin Kohn, Erik Walinda, Kenji Sugase, Daichi Morimoto, and Ulrich Scheler
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Fluid Flow and Transfer Processes ,Mechanics of Materials ,Mechanical Engineering ,Computational Mechanics ,Condensed Matter Physics - Abstract
Flow patterns including counter-flow and flow reversal effects have been studied by a combination of nuclear magnetic resonance flow imaging and numerical modeling using the finite volume method in the open-source computational fluid mechanics package OpenFOAM. Two cylindrical geometries have been used: In a concentric double-cylinder system the flow reversal under oscillatory rotation of the inner cylinder has been followed, and the time evolution of the flow reversal has been studied. We find extended periods of counter-rotating flow in the gap where fluid in the inner part of the gap follows the new direction of the rotor, while the outer part takes a longer time until the viscous forces transmit the reverted flow direction outwards. The radial position of the reversal of flow direction has been monitored as a function of the oscillation angle after the turning point. In the second cylindrical geometry, the rotating bob is placed off the center and a counter-rotating vortex is detected in the wider part of the gap. At constant viscosity and eccentricity, the position of the center of the vortex was found to depend on the rotation frequency of the bob. Qualitative and quantitative agreement between experiment and laminar (nonturbulent) flow simulations has been obtained for both steady-state flow using the Semi-Implicit Method for Pressure Linked Equations (SIMPLE) algorithm and time-dependent flow using the Pressure Implicit with Splitting of Operators (PISO) algorithm.
- Published
- 2022
22. Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by
- Author
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Yuka, Tanaka, Daichi, Morimoto, Erik, Walinda, Kenji, Sugase, and Masahiro, Shirakawa
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Kinetics ,Ubiquitin ,Ubiquitination ,Humans ,Oxygen Isotopes ,Mass Spectrometry - Abstract
Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied
- Published
- 2020
23. Resolving biomolecular motion and interactions by R2 and R1ρ relaxation dispersion NMR
- Author
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Erik Walinda, Kenji Sugase, and Daichi Morimoto
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0301 basic medicine ,Physics ,education.field_of_study ,Protein dynamics ,Population ,Relaxation (NMR) ,Pulse sequence ,010402 general chemistry ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,0104 chemical sciences ,Folding (chemistry) ,03 medical and health sciences ,030104 developmental biology ,Structural biology ,Chemical physics ,Excited state ,education ,Ground state ,Molecular Biology - Abstract
Among the tools of structural biology, NMR spectroscopy is unique in that it not only derives a static three-dimensional structure, but also provides an atomic-level description of the local fluctuations and global dynamics around this static structure. A battery of NMR experiments is now available to probe the motions of proteins and nucleic acids over the whole biologically relevant timescale from picoseconds to hours. Here we focus on one of these methods, relaxation dispersion, which resolves dynamics on the micro- to millisecond timescale. Key biological processes that occur on this timescale include enzymatic catalysis, ligand binding, and local folding. In other words, relaxation-dispersion-resolved dynamics are often closely related to the function of the molecule and therefore highly interesting to the structural biochemist. With an astounding sensitivity of ∼0.5%, the method detects low-population excited states that are invisible to any other biophysical method. The kinetics of the exchange between the ground state and excited states are quantified in the form of the underlying exchange rate, while structural information about the invisible excited state is obtained in the form of its chemical shift. Lastly, the population of the excited state can be derived. This diversity in the information that can be obtained makes relaxation dispersion an excellent method to study the detailed mechanisms of conformational transitions and molecular interactions. Here we describe the two branches of relaxation dispersion, R2 and R1ρ, discussing their applicability, similarities, and differences, as well as recent developments in pulse sequence design and data processing.
- Published
- 2018
24. Pinpoint analysis of a protein in slow exchange using F
- Author
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Mayu, Nishizawa, Erik, Walinda, Daichi, Morimoto, and Kenji, Sugase
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Kinetics ,Protein Domains ,Protein Conformation ,Ubiquitin ,Recombinant Fusion Proteins ,Sequestosome-1 Protein ,Humans ,Dimerization ,Magnetic Resonance Imaging ,Nuclear Magnetic Resonance, Biomolecular - Abstract
ZZ-exchange spectroscopy is widely used to study slow exchange processes in biomolecules, especially determination of exchange rates and assignment of minor peaks. However, if the exchange cross peaks overlap or the populations are skewed, kinetic analysis is hindered. In order to analyze slow exchange protein dynamics under such conditions, here we have developed a new method by combining ZZ-exchange and F
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- 2019
25. Conformational exchange in the potassium channel blocker ShK
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Nicola J. Baxter, Rodrigo A.V. Morales, Naoto Iwakawa, Kenji Sugase, Michael P. Williamson, Dorothy C.C. Wai, Nicholas J. Fowler, and Raymond S. Norton
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0301 basic medicine ,Population ,Hydrostatic pressure ,Molecular Conformation ,lcsh:Medicine ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Article ,Autoimmune Diseases ,03 medical and health sciences ,Molecular dynamics ,NMR spectroscopy ,Cnidarian Venoms ,medicine ,Potassium Channel Blockers ,Animals ,Humans ,Amino Acid Sequence ,education ,lcsh:Science ,education.field_of_study ,Multidisciplinary ,Kv1.3 Potassium Channel ,Stichodactyla helianthus ,biology ,Chemistry ,lcsh:R ,Disulfide bond ,Potassium channel blocker ,biology.organism_classification ,Potassium channel ,0104 chemical sciences ,030104 developmental biology ,Sea Anemones ,Helix ,Biophysics ,lcsh:Q ,Peptides ,Kv1.1 Potassium Channel ,medicine.drug - Abstract
ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21–24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.
- Published
- 2019
26. Expression, solubility monitoring, and purification of the co-folded LUBAC LTM domain by structure-guided tandem folding in autoinducing cultures
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Kenji Sugase, Tomoki Sorada, Erik Walinda, Daichi Morimoto, and Kazuhiro Iwai
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Protein Folding ,animal structures ,Protein Conformation ,Recombinant Fusion Proteins ,Ubiquitin-Protein Ligases ,Molecular Dynamics Simulation ,Green fluorescent protein ,Ubiquitin ,Humans ,Nuclear Magnetic Resonance, Biomolecular ,Ubiquitins ,Binding Sites ,biology ,Tandem ,Chemistry ,fungi ,Temperature ,Fusion protein ,Folding (chemistry) ,Solubility ,Heteronuclear molecule ,Chromatography, Gel ,biology.protein ,Biophysics ,Protein folding ,Primer (molecular biology) ,psychological phenomena and processes ,Protein Binding ,Signal Transduction ,Transcription Factors ,Biotechnology - Abstract
The l inear ubiquitin chain assembly complex t ethering m otif (LUBAC- LTM ) domain is composed of two different accessory LUBAC components (HOIL-1L and SHARPIN) but folds as a single globular domain. Targeted disruption of the intricate LTM-LTM interaction destabilizes LUBAC in lymphoma cells, thereby attenuating LUBAC stability, which highlights that targeting the interaction between the two LTM motifs is a promising strategy for the development of new agents against cancers that depend on LUBAC activity for their survival. To further screen for small-molecule inhibitors that can selectively disrupt the LTM-LTM interaction, it is necessary to obtain high-purity samples of the LTM domain. Ideally, such a sample would not contain any components other than the LTM itself, so that false positives (molecules binding to other parts of LUBAC) could be eliminated from the screening process. Here we report a simple strategy that enabled successful bacterial production of the isolated LUBAC LTM domain in high yield and at high purity. The strategy combines (1) structural analysis highlighting the possibility of tandem expression in the SHARPINL™ to HOIL-1LL™ direction; (2) bacterial expression downstream of EGFP to efficiently monitor expression and solubility; (3) gentle low-temperature folding using autoinduction. Formation of stably folded LTM was verified by size-exclusion chromatography and heteronuclear NMR spectroscopy. From 200-ml cultures sufficient quantities (~7 mg) of high-purity protein for structural studies could be obtained. The presented strategy will be beneficial for LUBAC LTM-based drug-screening efforts and likely serve as a useful primer for similar cases, i.e., whenever a smaller folded fragment is to be isolated from a larger protein complex for site-specific downstream applications.
- Published
- 2021
27. Glycyrrhizin Derivatives Suppress Cancer Chemoresistance by Inhibiting Progesterone Receptor Membrane Component 1
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Yasuaki Kabe, Masanori Noda, Makoto Suematsu, Hiroki Yamazaki, Tatsuya Yamamoto, Ayaka Kanai, Hiroaki Hayashi, Hiroshi Handa, Hirotoshi Tanaka, Hitoshi Tsugawa, Ryogo Furuhata, Miwa Hirai, Erisa Harada, Susumu Uchiyama, Takuya Kobayashi, Kenji Sugase, Ikko Koike, Kazue Hanadate, and Nobuji Yoshikawa
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glycyrrhizin ,0301 basic medicine ,Cancer Research ,Article ,03 medical and health sciences ,EGF receptor ,0302 clinical medicine ,Progesterone receptor ,medicine ,endocytosis ,Receptor ,PGRMC1 ,RC254-282 ,Chemistry ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,chemoresistance ,Cancer ,progesterone receptor membrane component 1 ,medicine.disease ,030104 developmental biology ,low-density lipoprotein ,Oncology ,Tumor progression ,030220 oncology & carcinogenesis ,glycyrrhizin derivatives ,LDL receptor ,Cancer cell ,Cancer research ,Erlotinib ,medicine.drug - Abstract
Simple Summary Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in cancer cells and enhances cancer proliferation and chemoresistance. It is therefore considered a potential target for cancer treatment. However, a chemical compound that directly regulates PGRMC1 has not been identified. Here, we showed that the natural active compound in licorice, glycyrrhizin (GL), directly binds to heme-dimerized PGRMC1 to inhibit PGRMC1-mediated EGF receptor (EGFR) activation in cancer cells. Chemical screening using GL derivatives revealed that the glucoside derivative glucoglycyrrhizin (GlucoGL) binds more potently to PGRMC1 and contributes to the suppression of PGRMC1-mediated cancer chemoresistance. This study provides the first evidence of chemical compounds that directly bind to PGRMC1 to inhibit its function, and the findings provide new insights for cancer treatments that target PGRMC1. Abstract Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in various cancer cells and contributes to tumor progression. We have previously shown that PGRMC1 forms a unique heme-stacking functional dimer to enhance EGF receptor (EGFR) activity required for cancer proliferation and chemoresistance, and the dimer dissociates by carbon monoxide to attenuate its biological actions. Here, we determined that glycyrrhizin (GL), which is conventionally used to ameliorate inflammation, specifically binds to heme-dimerized PGRMC1. Binding analyses using isothermal titration calorimetry revealed that some GL derivatives, including its glucoside-derivative (GlucoGL), bind to PGRMC1 potently, whereas its aglycone, glycyrrhetinic acid (GA), does not bind. GL and GlucoGL inhibit the interaction between PGRMC1 and EGFR, thereby suppressing EGFR-mediated signaling required for cancer progression. GL and GlucoGL significantly enhanced EGFR inhibitor erlotinib- or cisplatin (CDDP)-induced cell death in human colon cancer HCT116 cells. In addition, GL derivatives suppressed the intracellular uptake of low-density lipoprotein (LDL) by inhibiting the interaction between PGRMC1 and the LDL receptor (LDLR). Effects on other pathways cannot be excluded. Treatment with GlucoGL and CDDP significantly suppressed tumor growth following xenograft transplantation in mice. Collectively, this study indicates that GL derivatives are novel inhibitors of PGRMC1 that suppress cancer progression, and our findings provide new insights for cancer treatment.
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- 2021
28. High-Sensitivity Rheo-NMR Spectroscopy for Protein Studies
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Kenji Sugase, Ulrich Scheler, Daichi Morimoto, Masahiro Shirakawa, Akihiko Yamamoto, Yasushi Kawata, Erik Walinda, Mayu Nishizawa, and Naoto Iwakawa
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0301 basic medicine ,Magnetic Resonance Spectroscopy ,Cryogenic probes ,flow kinetics ,Fibril formation ,Nanotechnology ,Analytical Chemistry ,03 medical and health sciences ,Protein structure ,Rheology ,Protein structures ,Shear stress ,Humans ,human ,Sensitivity (control systems) ,Spectroscopy ,nuclear magnetic resonance spectroscopy ,Spectrometer ,Ubiquitin ,Chemistry ,Relaxation (NMR) ,Nuclear magnetic resonance spectroscopy ,Amyloid fibril formation ,NMR spectrometer ,Aggregate formation ,030104 developmental biology ,Structural deformation ,Rheological studies ,Biophysics - Abstract
Shear stress can induce structural deformation of proteins, which might result in aggregate formation. Rheo-NMR spectroscopy has the potential to monitor structural changes in proteins under shear stress at the atomic level; however, existing Rheo-NMR methodologies have insufficient sensitivity to probe protein structure and dynamics. Here we present a simple and versatile approach to Rheo-NMR, which maximizes sensitivity by using a spectrometer equipped with a cryogenic probe. As a result, the sensitivity of the instrument ranks highest among the Rheo-NMR spectrometers reported so far. We demonstrate that the newly developed Rheo-NMR instrument can acquire high-quality relaxation data for a protein under shear stress and can trace structural changes in a protein during fibril formation in real time. The described approach will facilitate rheological studies on protein structural deformation, thereby aiding a physical understanding of shear-induced amyloid fibril formation.
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- 2017
29. F 1 F 2-selective NMR spectroscopy
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Masahiro Shirakawa, Kenji Sugase, Erik Walinda, and Daichi Morimoto
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0301 basic medicine ,chemistry.chemical_classification ,Chemistry ,Chemical shift ,Biomolecule ,Analytical chemistry ,Nuclear magnetic resonance spectroscopy ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,03 medical and health sciences ,symbols.namesake ,030104 developmental biology ,Protein structure ,Fourier transform ,Dimensional reduction ,Frequency domain ,symbols ,Biological system ,Two-dimensional nuclear magnetic resonance spectroscopy ,Spectroscopy - Abstract
Fourier transform NMR spectroscopy has provided unprecedented insight into the structure, interaction and dynamic motion of proteins and nucleic acids. Conventional biomolecular NMR relies on the acquisition of three-dimensional and four-dimensional (4D) data matrices to establish correlations between chemical shifts in the frequency domains F 1, F 2, F 3 and F 1, F 2, F 3, F 4 respectively. While rich in information, these datasets require a substantial amount of acquisition time, are visually highly unintuitive, require expert knowledge to process, and sample dark and bright regions of the frequency domains equally. Here, we present an alternative approach to obtain multidimensional chemical shift correlations for biomolecules. This strategy focuses on one narrow frequency range, F 1 F 2, at a time and records the resulting F 3 F 4 correlation spectrum by two-dimensional NMR. As a result, only regions of the frequency domain that contain signals in F 1 F 2 (“bright regions”) are sampled. F 1 F 2 selection is achieved by Hartmann–Hahn cross-polarization using weak radio frequency fields. This approach reveals information equivalent to that of a conventional 4D experiment, while the dimensional reduction may shorten the total acquisition time and simplifies spectral processing, interpretation and comparative analysis. Potential applicability of the F 1 F 2-selective approach is illustrated by de novo assignment, structural and dynamics studies of ubiquitin and fatty-acid binding protein 4 (FABP4). Further extension of this concept may spawn new selective NMR experiments to aid studies of site-specific structural dynamics, protein–protein interactions and allosteric modulation of protein structure.
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- 2017
30. Practical considerations for investigation of protein conformational dynamics by 15N R 1ρ relaxation dispersion
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Kenji Sugase, Daichi Morimoto, Erik Walinda, and Masahiro Shirakawa
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0301 basic medicine ,Millisecond ,Chemistry ,Protein dynamics ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Spectral line ,0104 chemical sciences ,03 medical and health sciences ,030104 developmental biology ,Quality (physics) ,Chemical physics ,Computational chemistry ,Excited state ,Dispersion (optics) ,Relaxation (physics) ,Spectroscopy ,Excitation - Abstract
It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (“excited states”) has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by 15N R 1ρ relaxation dispersion. The schemes use selective Hartmann–Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713–721, 2005; Hansen et al. in J Am Chem Soc 131:3818–3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R 1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.
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- 2017
31. Backbone resonance assignments of monomeric SOD1 in dilute and crowded environments
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Erik Walinda, Masahiro Shirakawa, Naoto Iwakawa, Kenji Sugase, and Daichi Morimoto
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Models, Molecular ,0301 basic medicine ,SOD1 ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,Superoxide Dismutase-1 ,Structural Biology ,Humans ,Molecule ,Nuclear Magnetic Resonance, Biomolecular ,Protein secondary structure ,Chemistry ,Chemical shift ,nutritional and metabolic diseases ,nervous system diseases ,0104 chemical sciences ,Folding (chemistry) ,Crystallography ,030104 developmental biology ,Biophysics ,Muramidase ,Lysozyme ,Macromolecular crowding ,Intracellular - Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to movement disorders. In motor neurons of ALS patients, intracellular aggregates of superoxide dismutase 1 (SOD1) have often been observed. To elucidate the aggregation mechanism, it is important to analyze the folding equilibrium of SOD1 between folded and aggregation-prone unfolded states. However, in most cases, this folding equilibrium has been studied in dilute solution even though the aggregate formation occurs in a highly crowded intracellular environment. Indeed, a recent study reported that the folding stability of SOD1 decreased in an environment containing protein crowder molecules. To understand such a destabilization effect due to protein crowders, it is necessary to obtain more precise structural information on SOD1 in the presence of protein crowders. Here, we report the 1H, 13C, and 15N backbone resonance assignments of monomeric SOD1 in the absence and presence of the protein crowder lysozyme. The chemical shift differences caused by addition of lysozyme suggest that SOD1 associated with lysozyme via negatively charged surfaces. Based on the assigned chemical shifts, the presence of lysozyme has a limited influence on the secondary structure of SOD1. We anticipate that our assignments will provide an important basis for elucidation of the crowding-induced folding destabilization of SOD1.
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- 2016
32. The helical propensity of the extracellular loop is responsible for the substrate specificity of Fe(III)‐phytosiderophore transporters
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Yoshiko Murata, Erisa Harada, Kenji Sugase, and Kosuke Namba
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0301 basic medicine ,Circular dichroism ,Patch-Clamp Techniques ,Iron ,Biophysics ,Siderophores ,Plant Biology ,Peptide ,Zea mays ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,Structure-Activity Relationship ,Xenopus laevis ,03 medical and health sciences ,Protein Domains ,Species Specificity ,Gene Expression Regulation, Plant ,Structural Biology ,Research Letter ,Genetics ,Extracellular ,Animals ,Amino Acid Sequence ,Molecular Biology ,phytosiderophore ,Plant Proteins ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Chemistry ,Membrane Transport Proteins ,food and beverages ,Biological Transport ,Hordeum ,Transporter ,Cell Biology ,Recombinant Proteins ,Research Letters ,YS1 transporter ,Loop (topology) ,030104 developmental biology ,Membrane ,Oocytes ,α‐helical content ,Substrate specificity ,Hordeum vulgare ,Sequence Alignment - Abstract
Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter of Fe(III)‐phytosiderophores in barley that is responsible for iron acquisition from the soil. In contrast, maize Zea mays, yellow stripe 1 (ZmYS1) possesses broad substrate specificity. In this study, a quantitative evaluation of the transport activities of HvYS1 and ZmYS1 chimera proteins revealed that the seventh extracellular membrane loop is essential for substrate specificity. The loop peptides of both transporters were prepared and analysed by circular dichroism and NMR. The spectra revealed a higher propensity for α‐helical conformation of the HvYS1 loop peptide and a largely disordered structure for that of ZmYS1. These structural differences are potentially responsible for the substrate specificities of the transporters.
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- 2016
33. Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody
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Hiroko Tamura, Kouhei Tsumoto, Hiroki Akiba, Kenji Sugase, Jose M. M. Caaveiro, Masato Kiyoshi, and Saeko Yanaka
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Protein Conformation ,lcsh:Medicine ,Crystallography, X-Ray ,Article ,Epitope ,Substrate Specificity ,Protein structure ,Antigen ,Hen Egg Lysozyme ,Biophysical chemistry ,Concave surface ,Animals ,Humans ,lcsh:Science ,Nuclear Magnetic Resonance, Biomolecular ,X-ray crystallography ,Multidisciplinary ,Calorimetry, Differential Scanning ,biology ,Chemistry ,lcsh:R ,Proteins ,Substrate (chemistry) ,Single-Domain Antibodies ,Single-domain antibody ,Biophysics ,biology.protein ,Thermodynamics ,lcsh:Q ,Muramidase ,Antibody - Abstract
Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs.
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- 2019
34. Visualizing protein motion in Couette flow by all-atom molecular dynamics
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Masahiro Shirakawa, Kenji Sugase, Daichi Morimoto, Erik Walinda, and Ulrich Scheler
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Cytoplasm ,Protein Denaturation ,Materials science ,Magnetic Resonance Spectroscopy ,Biophysics ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Quantitative Biology::Subcellular Processes ,Physics::Fluid Dynamics ,Diffusion ,03 medical and health sciences ,Viscosity ,Motion ,Superoxide Dismutase-1 ,Protein Interaction Mapping ,Shear stress ,Humans ,Molecular Biology ,Couette flow ,030304 developmental biology ,Probability ,Shearing (physics) ,Cell Nucleus ,Quantitative Biology::Biomolecules ,0303 health sciences ,Dynamics (mechanics) ,Rotational diffusion ,Proteins ,Fluid mechanics ,Mechanics ,0104 chemical sciences ,Hydrodynamics ,Stress, Mechanical ,Shear flow ,Shear Strength - Abstract
In living cells, biomacromolecules are exposed to a highly crowded environment. The cytoplasm, the nucleus, and other organelles are highly viscous fluids that differ from dilute in vitro conditions. Viscosity, a measure of fluid internal friction, directly affects the forces that act on immersed macromolecules. Although active motion of this viscous fluid - cytoplasmic streaming - occurs in many plant and animal cells, the effect of fluid motion (flow) on biomolecules is rarely discussed. Recently NMR experiments that apply a shearing flow in situ have been used for protein studies. While these NMR experiments have succeeded in spectroscopically tracking protein aggregation in real time, they do not provide a visual picture of protein motion under shear. To fill this gap, here we have used molecular dynamics simulations to study the motion of three proteins of different size and shape in a simple shearing flow. The proteins exhibit a superposition of random diffusion and shear-flow-induced rotational motion. Random rotational diffusion dominates at lower shear stresses, whereas an active "rolling motion" along the axis of the applied flow occurs at higher shear stress. Even larger shear stresses perturb protein secondary structure elements resulting in local and global unfolding. Apart from shear-induced unfolding, our results imply that, in an ideal Couette flow field biomolecules undergo correlated motion, which should enhance the probability of inter-molecular interaction and aggregation. Connecting biomolecular simulation with experiments applying shear flow in situ appears to be a promising strategy to study protein alignment, deformation, and dynamics under shear.
- Published
- 2018
35. NMR resonance assignments of the NZF domain of mouse HOIL-1L free and bound to linear di-ubiquitin
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Kenji Sugase, Naoki Ishii, Masahiro Shirakawa, Kazuhiro Iwai, Naoto Iwakawa, Erik Walinda, and Daichi Morimoto
- Subjects
Stereochemistry ,030303 biophysics ,Biochemistry ,Protein Structure, Secondary ,Domain (software engineering) ,03 medical and health sciences ,Mice ,Chain (algebraic topology) ,Ubiquitin ,Protein Domains ,Structural Biology ,Side chain ,Animals ,Protein secondary structure ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,Zinc finger ,0303 health sciences ,biology ,Nitrogen Isotopes ,Chemistry ,Chemical shift ,Resonance (chemistry) ,biology.protein ,Protons ,Carrier Proteins ,Protein Binding - Abstract
Nuclear factor-κB (NF-κB) activation plays a central role in immunity and inflammation. In the canonical NF-κB activation pathway, linear polyubiquitin chains conjugated by the linear ubiquitin chain assembly complex (LUBAC) are specifically recognized by the Npl4 zinc finger (NZF) domain of heme-oxidized IRP2 ligase-1L (HOIL-1L). Recently, a crystal structure of the NZF domain in complex with linear di-ubiquitin has been reported; however, to understand the recognition mechanism in more detail, it is also necessary to investigate the structure and dynamics of the NZF domain in solution. In this study, we report the 1H, 13C, and 15N backbone and side chain resonance assignments of the NZF domain in the free form as well as the backbone resonance assignments of the NZF domain in the di-ubiquitin-bound form. Based on the assigned chemical shifts, we analyzed the secondary structure propensity, suggesting that the free form of the NZF domain forms secondary structure elements as observed in the di-ubiquitin-bound form. We expect that our data will provide an important basis for characterization of the free NZF domain and elucidation of the detailed recognition mechanism in solution.
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- 2018
36. Effects of Weak Nonspecific Interactions with ATP on Proteins.
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Mayu Nishizawa, Erik Walinda, Daichi Morimoto, Kohn, Benjamin, Scheler, Ulrich, Masahiro Shirakawa, and Kenji Sugase
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- 2021
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37. Dynamics of the Extended String-Like Interaction of TFIIE with the p62 Subunit of TFIIH
- Author
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Masahiko Okuda, Tadashi Komatsu, Junichi Higo, Kenji Sugase, Tsuyoshi Konuma, and Yoshifumi Nishimura
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0301 basic medicine ,Protein subunit ,Biophysics ,Molecular Dynamics Simulation ,010402 general chemistry ,Antiparallel (biochemistry) ,01 natural sciences ,Protein Structure, Secondary ,Transcription Factors, TFII ,03 medical and health sciences ,Molecular dynamics ,Protein Domains ,Humans ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Alanine ,Acidic Region ,Chemistry ,Proteins ,0104 chemical sciences ,Pleckstrin homology domain ,Crystallography ,030104 developmental biology ,Mutation ,Transcription factor II H ,Transcription factor II E ,Hydrophobic and Hydrophilic Interactions ,Transcription Factor TFIIH ,Algorithms ,Protein Binding - Abstract
General transcription factor II E (TFIIE) contains an acid-rich region (residues 378–393) in its α -subunit, comprising 13 acidic and two hydrophobic (Phe387 and Val390) residues. Upon binding to the p62 subunit of TFIIH, the acidic region adopts an extended string-like structure on the basic groove of the pleckstrin homology domain (PHD) of p62, and inserts Phe387 and Val390 into two shallow pockets in the groove. Here, we have examined the dynamics of this interaction by NMR and molecular dynamics (MD) simulations. Although alanine substitution of Phe387 and/or Val390 greatly reduced binding to PHD, the binding mode of the mutants was similar to that of the wild-type, as judged by the chemical-shift changes of the PHD. NMR relaxation dispersion profiles of the interaction exhibited large amplitudes for residues in the C-terminal half-string in the acidic region (Phe387, Glu388, Val390, Ala391, and Asp392), indicating a two-site binding mode: one corresponding to the final complex structure, and one to an off-pathway minor complex. To probe the off-pathway complex structure, an atomically detailed free-energy landscape of the binding mode was computed by all-atom multicanonical MD. The most thermodynamically stable cluster corresponded to the final complex structure. One of the next stable clusters was the off-pathway structure cluster, showing the reversed orientation of the C-terminal half-string on the PHD groove, as compared with the final structure. MD calculations elucidated that the C-terminal half-acidic-string forms encounter complexes mainly around the positive groove region with nearly two different orientations of the string, parallel and antiparallel to the final structure. Interestingly, the most encountered complexes exhibit a parallel-like orientation, suggesting that the string has a tendency to bind around the groove in the proper orientation with the aid of Phe387 and/or Val390 to proceed smoothly to the final complex structure.
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- 2016
38. Quantitative analysis of protein–ligand interactions by NMR
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Saeko Yanaka, Kenji Sugase, Tsuyoshi Konuma, and Ayako Furukawa
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0301 basic medicine ,Nuclear and High Energy Physics ,Chemistry ,Relaxation (NMR) ,Analytical chemistry ,Proteins ,Ligands ,Kinetic energy ,Ligand (biochemistry) ,Biochemistry ,Biophysical Phenomena ,Analytical Chemistry ,Dissociation constant ,Kinetics ,03 medical and health sciences ,030104 developmental biology ,Chemical physics ,Bound state ,Titration ,Dispersion (chemistry) ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Protein Binding ,Protein ligand - Abstract
Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used to analyze population-averaged NMR quantities. Essentially, to apply NMR successfully, both the type of experiment and equation to fit the data must be carefully and specifically chosen for the protein-ligand interaction under analysis. In this review, we first explain the exchange regimes and kinetic models of protein-ligand interactions, and then describe the NMR methods that quantitatively analyze these specific interactions.
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- 2016
39. Use of glass capillaries to suppress thermal convection in NMR tubes in diffusion measurements
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Tsuyoshi Konuma, Takashi Iwashita, Erisa Harada, Kenji Sugase, and Shoko Mori
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Convective heat transfer ,010405 organic chemistry ,Chemistry ,Capillary action ,Chemical shift ,Analytical chemistry ,NMR tube ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Spectral line ,0104 chemical sciences ,Physics::Fluid Dynamics ,Chemical physics ,Proton NMR ,General Materials Science ,Spectroscopy ,Heteronuclear single quantum coherence spectroscopy - Abstract
Diffusion ordered spectroscopy (DOSY) is used to determine the translational diffusion coefficients of molecules in solution. However, DOSY is highly susceptible to spurious spectral peaks resulting from thermal convection occurring in the NMR tube. Thermal convection therefore must be suppressed for accurate estimation of translational diffusion coefficients. In this study, we developed a new method to effectively suppress thermal convection using glass capillaries. A total of 6 to 18 capillaries (0.8-mm outer diameter) were inserted into a regular 5-mm NMR tube. The capillaries had minimal effect on magnetic field homogeneity and enabled us to obtain clean DOSY spectra of a mixture of small organic compounds. Moreover, the capillaries did not affect chemical shifts or signal intensities in two-dimensional heteronuclear single quantum coherence spectra. Capillaries are a simple and inexpensive means of suppressing thermal convection and thus can be used in a wide variety of DOSY experiments. Copyright © 2016 John Wiley & Sons, Ltd.
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- 2016
40. Overview of Relaxation Dispersion NMR Spectroscopy to Study Protein Dynamics and Protein-Ligand Interactions
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Kenji Sugase, Erik Walinda, and Daichi Morimoto
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0301 basic medicine ,Materials science ,Magnetic Resonance Spectroscopy ,Kinetics ,Molecular Dynamics Simulation ,010402 general chemistry ,Ligands ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,Structural Biology ,chemistry.chemical_classification ,Quantitative Biology::Biomolecules ,Protein dynamics ,Biomolecule ,Proteins ,Nuclear magnetic resonance spectroscopy ,0104 chemical sciences ,030104 developmental biology ,Heteronuclear molecule ,chemistry ,Chemical physics ,Relaxation (physics) ,Thermodynamics ,Dispersion (chemistry) ,Protein ligand - Abstract
Proteins and nucleic acids are central to all biological processes. NMR spectroscopy has proven to be excellent for studying the dynamics of these macromolecules over various timescales. Relaxation rates and heteronuclear nuclear Overhauser-effect values can resolve motion on pico- to nanosecond timescales, residual dipolar couplings provide information on submicro- to millisecond timescales, and even slower dynamics over seconds to hours can be resolved by hydrogen-exchange experiments. Relaxation dispersion experiments are especially valuable because they resolve motion on micro- to millisecond timescales, encompassing biomolecular motions associated with ligand binding, enzymatic catalysis, and domain-domain opening. These experiments provide structural, kinetic, and thermodynamic information on "invisible" excited conformational states. Relaxation dispersion can be applied not only to single biomolecules but also to protein-ligand complexes to study the kinetics and thermodynamics of association and dissociation. We review recent developments in relaxation dispersion methodology, outline the R1ρ relaxation dispersion experiment, and discuss application to biomolecular interactions. © 2018 by John Wiley & Sons, Inc.
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- 2018
41. Backbone and side-chain resonance assignments of the methyl-CpG-binding domain of MBD6 from Arabidopsis thaliana
- Author
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Arina Ono, Kenji Sugase, Naoto Iwakawa, Izuru Ohki, Masahiro Shirakawa, Daichi Morimoto, Yutaka Mahana, and Erik Walinda
- Subjects
Transposable element ,0303 health sciences ,Arabidopsis Proteins ,030303 biophysics ,Arabidopsis ,Computational biology ,Biochemistry ,MECP2 ,Methyl-CpG-binding domain ,DNA-Binding Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,Protein Domains ,Structural Biology ,DNA methylation ,Epigenetics ,Amino Acid Sequence ,Genomic imprinting ,Protein secondary structure ,Nuclear Magnetic Resonance, Biomolecular ,DNA ,030304 developmental biology - Abstract
Epigenetic regulation is essential to various biological phenomena such as cell differentiation and cancer. DNA methylation is one of the most important epigenetic signals, as it is directly involved in gene silencing of transposable elements, genomic imprinting, and chromosome X inactivation. To mediate these processes, methyl-CpG-binding domain (MBD) proteins recognize specific signals encoded in the form of DNA methylation patterns. AtMBD6, one of the 12 MBD proteins in Arabidopsis thaliana, shares a high sequential homology in the MBD domain with mammalian MBD proteins, but a detailed characterization of its structural and functional properties remains elusive. Here, we report the 1H, 13C, and 15N resonance assignments of the isolated MBD domain of AtMBD6. Analysis of the chemical shift data implied that the MBD domain of AtMBD6 has a secondary structure similar to that of mammalian MeCP2, while the β-strands β1 and β3 of AtMBD6 were found to be longer than those of MeCP2. The structural differences provide insight into the different recognition mechanisms of methylated DNA by plant and mammalian MBDs. The assignments reported here will aid further analyses such as titration experiments and three-dimensional structure determination using NMR to yield a detailed characterization of the interaction between AtMBD6 and methylated DNAs.
- Published
- 2018
42. Isolation and characterization of a minimal building block of polyubiquitin fibrils
- Author
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Mayo Shinke, Daichi Morimoto, Erik Walinda, Kenji Sugase, and Masahiro Shirakawa
- Subjects
0301 basic medicine ,Amyloid ,Protein Conformation ,lcsh:Medicine ,macromolecular substances ,Protein aggregation ,Fibril ,environment and public health ,Article ,03 medical and health sciences ,Ubiquitin ,Molecule ,Humans ,Disulfides ,lcsh:Science ,Polyubiquitin ,Multidisciplinary ,biology ,Chemistry ,lcsh:R ,Disulfide bond ,Characterization (materials science) ,030104 developmental biology ,Biophysics ,biology.protein ,lcsh:Q ,Protein Multimerization ,Intracellular - Abstract
As a posttranslational modifier, polyubiquitin is involved in the regulation of diverse intracellular processes; however, it is also found in pathological protein aggregates associated with Alzheimer’s disease and other neurodegenerative disorders. We previously observed that various types of polyubiquitin can form amyloid-like fibrils; however, the structural properties of these polyubiquitin fibrils have not been examined at an atomic level. Here we demonstrate that a soluble intermediate species can be extracted from disulfide-conjugated diubiquitin fibrils after cleaving the disulfide bonds in the fibrils. This newly discovered molecule is structurally and physicochemically distinguishable from native ubiquitin. In addition, it is thermodynamically metastable, as demonstrated by real-time NMR measurements. Collectively, our results suggest that the fibril-derived molecule is a minimal building block of polyubiquitin fibrils that reflects their structural and physicochemical properties.
- Published
- 2017
43. Elucidating Functional Dynamics by R 1ρ and R 2 Relaxation Dispersion NMR Spectroscopy
- Author
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Kenji Sugase and Erik Walinda
- Subjects
Quantitative Biology::Biomolecules ,Materials science ,Heteronuclear molecule ,Chemical physics ,Protein dynamics ,Relaxation (NMR) ,Pulse sequence ,Transverse relaxation-optimized spectroscopy ,Context (language use) ,Nuclear magnetic resonance spectroscopy ,Dispersion (chemistry) - Abstract
NMR spectroscopy is the method of choice to measure protein and nucleic acid dynamics on a variety of timescales. Picosecond to nanosecond dynamics can be precisely probed by quantifying R 1 and R 2 relaxation rates and heteronuclear NOE values, whereas residual dipolar couplings (RDCs) are sensitive to motion on a wide range of timescales from submicrosecond to milliseconds. Even slower dynamics can be assessed by hydrogen exchange experiments. In a biochemical context, relaxation dispersion NMR spectroscopy is particularly valuable, because it reports on the biologically important timescale from micro- to milliseconds, encompassing the conformational rearrangements of ligand binding, enzymatic reactions, and base pair transitions. From relaxation dispersion measurements, it is possible to obtain structural, kinetic, and thermodynamic information about energetically excited conformational minor states beyond the ground state structure. Here, we review the two methods of R 1ρ and R 2 relaxation dispersion, focusing on recent developments in pulse sequence design and data processing techniques, as well as applications of the methods to resolve protein–protein interactions.
- Published
- 2017
44. Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy
- Author
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Erisa Harada, Tsuyoshi Konuma, and Kenji Sugase
- Subjects
Models, Molecular ,Fourier Analysis ,Heme binding ,Protein Conformation ,Chemistry ,Protein dynamics ,Relaxation (NMR) ,Nuclear magnetic resonance spectroscopy ,Intrinsically disordered proteins ,Biochemistry ,Rats ,Solutions ,Nuclear magnetic resonance ,Protein structure ,Chemical physics ,Heme Oxygenase (Decyclizing) ,Dispersion (optics) ,Animals ,Transverse relaxation-optimized spectroscopy ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.
- Published
- 2015
45. Dynamic changes in CCAN organization through CENP-C during cell-cycle progression
- Author
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Kenji Sugase, Ayako Furukawa, Hitoshi Kurumizaka, Harsh Nagpal, Akihisa Osakabe, Tetsuya Hori, and Tatsuo Fukagawa
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Kinetochore ,Chromosomal Proteins, Non-Histone ,Kinetochore assembly ,Cell Cycle ,Centromere ,Mitosis ,Cell Biology ,macromolecular substances ,Articles ,Cell cycle ,Biology ,Chromatin ,Cell biology ,Nucleosomes ,Protein Structure, Tertiary ,Chromosome segregation ,Animals ,Centromere localization ,Kinetochores ,Molecular Biology ,Chickens ,Interphase - Abstract
Dynamic changes in CCAN organization during progression of the cell cycle are examined in chicken DT40 cells. CENP-C166-324 is sufficient for interphase centromere localization through association with CENP-L-N, and CENP-C643-864 is essential for mitotic centromere localization through binding to CENP-A nucleosomes., The kinetochore is a crucial structure for faithful chromosome segregation during mitosis and is formed in the centromeric region of each chromosome. The 16-subunit protein complex known as the constitutive centromere-associated network (CCAN) forms the foundation for kinetochore assembly on the centromeric chromatin. Although the CCAN can be divided into several subcomplexes, it remains unclear how CCAN proteins are organized to form the functional kinetochore. In particular, this organization may vary as the cell cycle progresses. To address this, we analyzed the relationship of centromeric protein (CENP)-C with the CENP-H complex during progression of the cell cycle. We find that the middle portion of chicken CENP-C (CENP-C166–324) is sufficient for centromere localization during interphase, potentially through association with the CENP-L-N complex. The C-terminus of CENP-C (CENP-C601–864) is essential for centromere localization during mitosis, through binding to CENP-A nucleosomes, independent of the CENP-H complex. On the basis of these results, we propose that CCAN organization changes dynamically during progression of the cell cycle.
- Published
- 2015
46. Real-Time Observation of the Interaction between Thioflavin T and an Amyloid Protein by Using High-Sensitivity Rheo-NMR
- Author
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Kenji Sugase, Naoto Iwakawa, Erik Walinda, Masahiro Shirakawa, Daichi Morimoto, and Yasushi Kawata
- Subjects
0301 basic medicine ,Amyloid ,Magnetic Resonance Spectroscopy ,Fluorescence assay ,Amyloidogenic Proteins ,Plasma protein binding ,macromolecular substances ,Rheo-NMR ,Fibril ,Article ,Catalysis ,lcsh:Chemistry ,Inorganic Chemistry ,amyloid fibrils ,03 medical and health sciences ,chemistry.chemical_compound ,thioflavin T ,molecular interactions ,real-time observation ,SOD1 ,Superoxide Dismutase-1 ,Benzothiazoles ,Physical and Theoretical Chemistry ,lcsh:QH301-705.5 ,Molecular Biology ,Spectroscopy ,Chemistry ,Organic Chemistry ,General Medicine ,Nuclear magnetic resonance spectroscopy ,Amyloid fibril ,Fluorescence ,Computer Science Applications ,Thiazoles ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,Biochemistry ,Thioflavin ,Protein Binding - Abstract
Amyloid fibril formation is associated with numerous neurodegenerative diseases. To elucidate the mechanism of fibril formation, the thioflavin T (ThT) fluorescence assay is widely used. ThT is a fluorescent dye that selectively binds to amyloid fibrils and exhibits fluorescence enhancement, which enables quantitative analysis of the fibril formation process. However, the detailed binding mechanism has remained unclear. Here we acquire real-time profiles of fibril formation of superoxide dismutase 1 (SOD1) using high-sensitivity Rheo-NMR spectroscopy and detect weak and strong interactions between ThT and SOD1 fibrils in a time-dependent manner. Real-time information on the interaction between ThT and fibrils will contribute to the understanding of the binding mechanism of ThT to fibrils. In addition, our method provides an alternative way to analyze fibril formation.
- Published
- 2017
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47. Elucidation of potential sites for antibody engineering by fluctuation editing
- Author
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Saeko Yanaka, Kenji Sugase, Kouhei Tsumoto, and Yoshitaka Moriwaki
- Subjects
Models, Molecular ,0301 basic medicine ,Protein Conformation ,medicine.drug_class ,Mutant ,Antibody Affinity ,Quantitative Structure-Activity Relationship ,lcsh:Medicine ,Complementarity determining region ,Biology ,Protein Engineering ,Monoclonal antibody ,Article ,Immunoglobulin Fab Fragments ,03 medical and health sciences ,Protein structure ,Antigen ,medicine ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Antigens ,Binding site ,lcsh:Science ,Binding Sites ,Multidisciplinary ,030102 biochemistry & molecular biology ,Protein Stability ,Point mutation ,lcsh:R ,Antibodies, Monoclonal ,Protein engineering ,Complementarity Determining Regions ,030104 developmental biology ,Mutation ,Immunology ,Biophysics ,Thermodynamics ,lcsh:Q - Abstract
Target-specific monoclonal antibodies can be routinely acquired, but the sequences of naturally acquired antibodies are not always affinity-matured and methods that increase antigen affinity are desirable. Most biophysical studies have focused on the complementary determining region (CDR), which directly contacts the antigen; however, it remains difficult to increase the affinity as much as desired. While strategies to alter the CDR to increase antibody affinity are abundant, those that target non-CDR regions are scarce. Here we describe a new method, designated fluctuation editing, which identifies potential mutation sites and engineers a high-affinity antibody based on conformational fluctuations observed by NMR relaxation dispersion. Our data show that relaxation dispersion detects important fluctuating residues that are not located in the CDR and that increase antigen–antibody affinity by point mutation. The affinity-increased mutants are shown to fluctuate less in their free form and to form a more packed structure in their antigen-bound form.
- Published
- 2017
48. Exploration of the Conformational Dynamics of Major Histocompatibility Complex Molecules
- Author
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Kenji Sugase and Saeko Yanaka
- Subjects
lcsh:Immunologic diseases. Allergy ,0301 basic medicine ,Mini Review ,conformational dynamics ,Immunology ,Peptide ,chemical and pharmacologic phenomena ,Major histocompatibility complex ,03 medical and health sciences ,0302 clinical medicine ,Immunology and Allergy ,Molecule ,Peptide sequence ,chemistry.chemical_classification ,biology ,Chemistry ,transient induced-fit model ,T-cell receptor ,Dynamics (mechanics) ,stability ,Acquired immune system ,major histocompatibility complex ,nuclear magnetic resonance ,030104 developmental biology ,Order (biology) ,biology.protein ,Biophysics ,relaxation dispersion ,lcsh:RC581-607 ,030215 immunology - Abstract
Major histocompatibility complex (MHC) molecules are loaded with a wide variety of self- and non-self-peptides in their binding grooves and present these to T cell receptors (TCRs) in order to activate the adaptive immune system. A large number of crystal structures of different MHC alleles with different bound peptides have been determined, and they have been found to be quite similar to one another regardless of the bound peptide sequence. The structures do not change markedly even when forming complexes with TCRs. Nonetheless, the degree of TCR activation does differ markedly depending on the peptide presented by the MHC. Recent structural studies in solution rather than as crystals have suggested that the conformational dynamics of MHC molecules may be responsible for the MHC stability differences. Furthermore, it was shown that the conformational dynamics of MHC molecules is important for peptide loading and presentation to TCR. Here, we describe the static and dynamic structures of MHC molecules and appropriate methods to analyze them. We focus particularly on nuclear magnetic resonance (NMR), one of the most powerful tools to study dynamic properties of proteins. The number of such studies in the literature is limited, but in this review, we show that NMR is valuable for elucidating the structural dynamics of MHC molecules.
- Published
- 2017
49. Biological and Physicochemical Functions of Ubiquitylation Revealed by Synthetic Chemistry Approaches
- Author
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Kenji Sugase, Erik Walinda, Masahiro Shirakawa, and Daichi Morimoto
- Subjects
0301 basic medicine ,Review ,010402 general chemistry ,01 natural sciences ,Chemical synthesis ,Catalysis ,chemical ubiquitylation ,Inorganic Chemistry ,lcsh:Chemistry ,03 medical and health sciences ,Ubiquitin ,ubiquitin ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,lcsh:QH301-705.5 ,Spectroscopy ,biology ,Intracellular protein ,Chemistry ,Organic Chemistry ,Ubiquitination ,Proteins ,General Medicine ,Enzymatic synthesis ,0104 chemical sciences ,Computer Science Applications ,030104 developmental biology ,Biochemistry ,post-translational modification ,lcsh:Biology (General) ,lcsh:QD1-999 ,Posttranslational modification ,biology.protein ,site-directed conjugation ,Protein Processing, Post-Translational - Abstract
Most intracellular proteins are subjected to post-translational modification by ubiquitin. Accordingly, it is of fundamental importance to investigate the biological and physicochemical effects of ubiquitylation on substrate proteins. However, preparation of ubiquitylated proteins by an enzymatic synthesis bears limitations in terms of yield and site-specificity. Recently established chemical ubiquitylation methodologies can overcome these problems and provide a new understanding of ubiquitylation. Herein we describe the recent chemical ubiquitylation procedures with a focus on the effects of ubiquitylation on target proteins revealed by the synthetic approach.
- Published
- 2017
50. F
- Author
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Erik, Walinda, Daichi, Morimoto, Masahiro, Shirakawa, and Kenji, Sugase
- Subjects
Magnetic Resonance Spectroscopy ,Fourier Analysis ,Protein Conformation ,Ubiquitin ,Isotope Labeling ,Humans ,Proteins ,Fatty Acid-Binding Proteins - Abstract
Fourier transform NMR spectroscopy has provided unprecedented insight into the structure, interaction and dynamic motion of proteins and nucleic acids. Conventional biomolecular NMR relies on the acquisition of three-dimensional and four-dimensional (4D) data matrices to establish correlations between chemical shifts in the frequency domains F
- Published
- 2017
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