Publisher：Cold Spring Harbor Laboratory  

ABSTRACT

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. Here, we present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a widely conserved auxiliary factor. NMR and site-specific crosslinking experiments revealed two distinct sites within the intrinsically disordered N-terminus of Sfr1 that cooperatively bind to Rad51. Although disruption of this binding severely impaired Rad51 stimulation in vitro, interaction mutants did not show any defects in DNA repair. Unexpectedly, in the absence of the Rad51 paralogs Rad55-Rad57, which constitute another auxiliary factor complex, these interaction mutants were unable to promote DNA repair. Our findings provide molecular insights into Rad51 stimulation by Swi5-Sfr1 and suggest that, rather than functioning in an independent subpathway of HR as was previously proposed, Rad55-Rad57 facilitates the recruitment of Swi5-Sfr1 to Rad51.

DOI： 10.1101/738179

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

alpha-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid (AMPA) ionotropic glutamate receptors mediate fast excitatory neurotransmission in the central nervous system, and their dysfunction is associated with neurological diseases. Glutamate binding to ligand-binding domains (LBDs) of AMPA receptors induces channel opening in the transmembrane domains of the receptors. The T686A mutation reduces glutamate efficacy so that the glutamate behaves as a partial agonist. The crystal structures of wild-type and mutant LBDs are very similar and cannot account for the observed behavior. To elucidate the molecular mechanism inducing partial agonism of the T686A mutant, we computed the free-energy landscapes governing GIuA2 LBD closure using replica-exchange umbrella sampling simulations. A semiclosed state, not observed in crystal structures, appears in the mutant during simulation. In this state, the LBD cleft opens slightly because of breaking of interlobe hydrogen bonds, reducing the efficiency of channel opening. The energy difference between the LBD closed and semiclosed states is small, and transitions between the two states would occur by thermal fluctuations. Evidently, glutamate binding to the T686A mutant induces a population shift from a closed to a semiclosed state, explaining the partial agonism in the AMPA receptor.

DOI： 10.1016/j.bpj.2018.11.3122

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Sphingolipids such as ceramide are important constituents of cell membranes. The ceramide transfer protein (CERT) moves ceramide from the endoplasmic reticulum to the Golgi apparatus in a nonvesicular manner. Hyperphosphorylation of the serine-repeat motif (SRM) adjacent to the pleckstrin homology (PH) domain of CERT down-regulates the inter-organelle ceramide transport function of CERT. However, the mechanistic details of this down-regulation remain elusive. Using solution NMR and binding assays, we herein show that a hyperphosphorylation-mimetic CERT variant in which 10 serine/threonine residues of SRM had been replaced with glutamate residues (the 10E variant) displays an intramolecular interaction between SRM and positively charged regions of the PH domain, which are involved in the binding of this domain to phosphatidylinositol 4-monophosphate (PI4P). Of note, the binding of the PH domain to PI4P-embedded membranes was attenuated by the SRM 10E substitutions in cell-free assays. Moreover, the 10E substitutions reduced the Golgi-targeting activity of the PH-SRM construct in living cells. These results indicate that hyperphosphorylated SRM directly interacts with the surface of the PH domain in an intramolecular manner, thereby decreasing the PI4P-binding activity of the PH domain. In light of these findings, we propose that the hyperphosphorylation of SRM may trigger the dissociation of CERT from the Golgi apparatus, resulting in a functionally less active conformation of CERT.

DOI： 10.1074/jbc.RA118.002465

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense 1H–13C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective 13C incorporation by the P. pastoris expression system.

DOI： 10.1007/s10858-018-0192-3

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/febs.14397

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.14397

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

DOI： 10.1038/NCHEM.2554

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Numerous small organic compounds exist in equilibrium among monomers, soluble oligomers, and insoluble aggregates in aqueous solution. Compound aggregation is a major reason for false positives in drug screening, and even soluble oligomers can interfere with structural and biochemical analyses. However, an efficient way to manage the equilibrium of aggregation-prone compounds, especially those involved with soluble oligomers, has not been established. In this study, solution NMR spectroscopy was used as a suitable technique to detect compound oligomers in equilibrium, and it was demonstrated that cosolubilization of nondetergent sulfobetaines (NDSBs) can largely suppress compound oligomerization and aggregation by shifting the equilibrium toward the monomers. The rotational correlation time was obtained from the ratio of the selective and nonselective longitudinal NMR relaxation times, which directly and quantitatively reflected the apparent sizes of the compounds in the equilibrium. The rotational correlation time of the aggregation-prone compound SKF86002 (1mM) was substantially reduced from 0.31 to 0.23ns by cosolubilization of 100mM NDSB195. NDSB cosolubilization allowed us to perform successful structural and biochemical experiments with substantially fewer artifacts, which represents a strategy to directly resolve the problematic oligomerization and aggregation of compounds.

DOI： 10.1002/cmdc.201500057

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LmrR is a multidrug transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. However, the molecular mechanism by which LmrR binds to structurally unrelated compounds and is released from the promoter region remains largely unknown. Here, we structurally and dynamically characterized LmrR in the apo, compound-bound and promoter-bound states. The compound-binding site of LmrR exhibits ps-mu s dynamics in the apo state, and compound ligation shifts the preexisting conformational equilibrium to varying extents to achieve multidrug recognition. Meanwhile, the compound binding induces redistribution of ps-ns dynamics to the allosteric sites, which entropically favors the high-affinity recognition. Furthermore, the reciprocal compound/promoter binding by LmrR is achieved by the incompatible conformational ensembles between the compound-and promoter-bound states. Collectively, the data show how LmrR can dynamically exert its functions through promiscuous multi-target interactions, in a manner that cannot be understood by a static structural view.

DOI： 10.1038/srep06922

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Mitogen-activated protein kinases (MAPKs) are essential to intracellular signal transduction. MAPKs anchor their pathway-specific substrates through so-called 'docking interactions' at locations distal from the active site. Docking interactions ensure efficient substrate recognition, but their contribution to the kinase reaction itself remains unclear. Herein, we use solution NMR to analyze the interaction between dually phosphorylated, active human p38 alpha and the C-terminal fragments of its substrate MK2. p38 alpha phosphorylation and ATP loading collaboratively induce the active conformation; subsequently, p38 alpha accommodates MK2 phosphoacceptor residues in its active site. The docking interaction enhances binding of ATP and the phosphoacceptor to p38 alpha, accelerating the phosphotransfer reaction. Thus, the docking interaction enhances p38 alpha's enzymatic activity toward pathway-specific substrates allosterically as well as by the anchor effect. These findings clarify how MAPK cascades are organized in cells, even under ATP-depleted conditions often associated with environmental stress.

DOI： 10.1038/nsmb.2861

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Structural information about the target-compound complex is invaluable in the early stage of drug discovery. In particular, it is important to know into which part of the initial compound additional interaction sites could be introduced to improve its affinity. Herein, we demonstrate that the affinity of a small-molecule inhibitor for its target protein could be successfully improved by the constructive introduction of the interaction mode of a competitive peptide. The strategy involved the discrimination of overlapping and non-overlapping peptide-compound pharmacophores by the use of a ligand-based NMR spectroscopic approach, INPHARMA. The obtained results enabled the design of a new compound with improved affinity for the platelet receptor glycoproteinVI (GPVI). The approach proposed herein efficiently combines the advantages of compounds and peptides for the development of higher-affinity druglike ligands.

DOI： 10.1002/anie.201310749

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Other Link： http://orcid.org/0000-0002-6227-4627

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The production of stable isotope-labeled proteins is critical in structural analyses of large molecular weight proteins using NMR. Although prokaryotic expression systems using Escherichia coli have been widely used for this purpose, yeast strains have also been useful for the expression of functional eukaryotic proteins. Recently, we reported a cost-effective stable isotope-labeled protein expression using the hemiascomycete yeast Kluyveromyces lactis (K. lactis), which allow us to express exogenous proteins at costs comparable to prokaryotic expression systems. Here, we report the successful production of highly deuterated (>90%) protein in the K. lactis system. We also examined the methyl-selective H-1, C-13-labeling of Ile, Leu, and Val residues using commonly used amino acid precursors. The efficiency of H-1-C-13-incorporation varied significantly based on the amino acid. Although a high level of H-1-C-13-incorporation was observed for the Ile delta 1 position, H-1, C-13-labeling rates of Val and Leu methyl groups were limited due to the mitochondrial localization of enzymes involved in amino acid biosynthesis and the lack of transporters for alpha-ketoisovalerate in the mitochondrial membrane. In line with this notion, the co-expression with branched-chain-amino-acid aminotransferase in the cytosol significantly improved the incorporation rates of amino acid precursors. Although it would be less cost-effective, addition of C-13-labeled valine can circumvent problems associated with precursors and achieve high level H-1, C-13-labeling of Val and Leu. Taken together, the K. lactis system would be a good alternative for expressing large eukaryotic proteins that need deuteration and/or the methyl-selective H-1, C-13-labeling for the sensitive detection of NMR resonances.

DOI： 10.1007/s10858-013-9789-8

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

In this study, we developed an assignment-free approach for rapid identification of ligand-binding sites in target proteins by using NMR With a sophisticated cell-free stable isotope-labeling procedure that introduces N-15- or C-13-labels to specific atoms of target proteins, this approach requires only a single series of ligand titrations with labeled targets. Using titration data, ligand-binding sites in the target protein can be identified without time-consuming assignment procedures. We demonstrated the feasibility of this approach by using structurally well-characterized interactions between mitogen-activated protein (MAP) kinase p38 alpha and its inhibitor 2-amino-3-benzyloxypyridine. Furthermore, we confirmed the recently proposed fatty acid binding to p38 alpha and confirmed the fatty acid-binding site in the MAP kinase insert region.

DOI： 10.1021/jm4014357

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：化学同人  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the process relies on the ceramide trafficking protein (CERT), which contains pleckstrin homology (PH) and StAR-related lipid transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate (PtdIns(4)P), a characteristic phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide by CERT. In this study, we determined the three-dimensional structure of the CERT PH domain by using solution NMR techniques. The structure revealed the presence of a characteristic basic groove near the canonical PtdIns(4) P recognition site. An extensive interaction study using NMRand other biophysical techniques revealed that the basic groove coordinates the CERT PH domain for efficient PtdIns(4)P recognition and localization in the Golgi apparatus. The notion was also supported by Golgi mislocalization of the CERT mutants in living cells. The distinctive binding modes reflect the functions of PH domains, as the basic groove is conserved only in thePHdomains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity.

DOI： 10.1074/jbc.M112.367730

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Asialoglycoprotein receptor (ASGP-R) is an endocytic C-type lectin receptor in hepatocytes that clears plasma glycoconjugates containing a terminal galactose or N-acetylgalactosamine. The carbohydrate recognition domain (CRD) of ASGP-R has three Ca2+ binding sites (sites 1, 2 and 3), with Ca2+ at site 2 being directly involved in ligand binding. Following endocytosis, the ligands are released from ASGP-R in endosomes to allow receptor recycling to the cell membrane. Although dissociation of the receptorligand complex is mediated by the acidic environment within the mature endosomes, many of these complexes also dissociate in the early time of endocytosis, where pH is approximately neutral. To investigate the mechanism of ligand release from ASGP-R in early endosomes, we examined the binding mode of Ca2+ and ligands to ASGP-R CRD by NMR. We demonstrate that sites 1 and 2 of ASGP-R are high affinity Ca2+ binding sites, site 3 is low affinity, and that Ca2+ ions bind to sites 1 and 2 cooperatively. The pH and Ca2+ concentration dependences of Ca2+ binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that the cooperative binding mode of Ca2+ makes it possible for ASGP-R to be more sensitive to Ca2+ concentrations in early endosomes, and plays an important role in the efficient release of ligand from ASGP-R. In our proposed mechanism, ASGP-R can rapidly release Ca2+ and its ligand even at nearly neutral pH. Sequence comparisons of endocytic C-type lectin receptors suggest that this mechanism is common in their family.

DOI： 10.1111/j.1742-4658.2012.08643.x

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DOI： 10.1007/978-1-61779-480-3_2

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

DOI： 10.1002/anie.201104905

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DOI： 10.2210/pdb2rsg/pdb

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Other Link： http://orcid.org/0000-0002-6227-4627

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

VHH is the binding domain of the IgG heavy chain. Some VHHs have an extremely long CDR3 that contributes to antigen binding. We studied the antigen binding ability of CDR3 by grafting a CDR3 from an antigen-binding VHH onto a nonbinding VHH. cAb-CA05-(1RI8), the CDR3-grafted VHH, had an antigen-binding ability. To find a human scaffold protein acceptable for VHH CDR3 grafting, we focused on the conserved structure of VHH, especially the N-terminal and C-terminal amino acid residues of the CDR3 loop and the Cys residue of CDR1. Human origin protein structures with the same orientation were searched in PDB and ubiquitin was selected. Ubi-(1RI8), the CDR3-grafted ubiquitin, had antigen-binding ability, though the affinity was relatively low compared to cAb-CA05-(1RI8). The thermodynamic parameters of Ubi-(1RI8) binding to HEWL were different from cAb-CA05-(1RI8). Hydrogen-deuterium exchange experiments showed decreased stability around the CDR3 grafting region of Ubi-(1RI8), which might explain the decreased antigen-binding ability and the differences in thermodynamic properties. We concluded that the orientation of the CDR3 sequence of Ubi-(1RI8) could not be reconstructed correctly.

DOI： 10.1002/pro.734

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DOI： 10.1016/j.jmgm.2011.08.002

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 20 titration experiments of a single protein sample, labeled with a unique combination of an N-15-labeled amino acid and several amino acids C-13-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a omega(1)-TROSY-type backbone H-1-N-15 and aromatic H-1-C-13 shift correlations together with single quantum methyl H-1-C-13 shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jsb.2011.04.001

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DOI： 10.1007/s10858-010-9456-2

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DOI： 10.1007/s10858-010-9402-3

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DOI： 10.1016/j.ab.2009.12.031

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

GPVI is a key receptor For collagen-induced platelet activation. Loss or inhibition of GPVI causes only mildly prolonged bleeding times but prevents arterial thrombus formation in animal models. Therefore, GPVI is considered to be a potent target molecule for therapy of thrombotic diseases. Recently, it was reported that the AT(1)-receptor antagonist losartan (DuP-753) and EXP3179 inhibit platelet adhesion and aggregation via GPVI. However, it is still not clear how losartan is associated with inhibition of binding between GPVI and collagen at the molecular level. Here, we show by NMR that losartan directly interacts with the hydrophobic region consisting of strands C' and E in the N-terminal Ig-like domain of GPVI. A reliable GPVI-losartan complex model is presented by using a combination of NMR data and in silico tools. These data indicated that the phenyl group with the tetrazole ring in losartan plays a Crucial role in the interaction with GPVI.

DOI： 10.1021/jm901534d

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Insulin initiates metabolic control by binding to the insulin receptor (IR) on target cells. Kinetic and mutational analyses have revealed two binding sites on the insulin molecule and the residues that compose them. However, direct determination of the insulin-IR interface is required to distinguish those residues that contribute to receptor binding from those required for structural stability. Here, we Successfully characterized one binding Site Using the nuclear magnetic resonance (NMR) transferred cross-saturation method, which can directly determine the binding interface of a large protein-protein complex. The results showed that this binding site contained three residues that have not been identified previously by mutational analyses. On the basis of the structure of the contact site, we also identified a molecule that can displace insulin from the IR. In addition, we discuss the mode of interaction between insulin and its receptor relative to the NMR analyses.

DOI： 10.1021/jm901099v

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DOI： 10.1007/s10858-009-9377-0

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The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 β-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M109.024851

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DOI： 10.1074/jbc.M109.001149

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Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single-molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high-throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.

DOI： 10.1002/pro.92

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Glycoprotein VI (GPVI) is a major collagen receptor on the platelet surface that recognizes the glycine-proline-hydroxyproline (GPO) sequence in the collagen molecule and plays a crucial role in thrombus formation. Inhibitors that block the interaction of GPVI with collagen have potential for use as antithrombotic drugs. For low molecular weight drug design for GPVI, it is essential to obtain precise structural and interaction information about GPVI-binding ligands. However, experimentally obtained structural and interaction information of small ligands, such as peptides, in the GPVI-bound state has not been reported. In this study, by screening a phage-displayed peptide library, we discovered a novel peptide ligand (pep-10L; YSDTDWLYFSTS) without any similarities to the sequence of collagen that inhibits GPVI-GPO binding. Systematic Ala scanning in surface plasmon resonance experiments and a saturation transfer difference NMR experiment revealed that Trp(6), Leu(7), Phe(9), and Ser(10) residues in the pep-10L peptide interacted with GPVI. Furthermore, the GPVI-bound conformation of the pep-10L peptide was determined using transferred nuclear Overhauser effect analysis. The obtained structure has revealed that the central part of pep-10L (Asp(5)-Phe(9)) has a helical conformation, the side chains of Trp(6), Leu(7), and Phe(9) form a hydrophobic side in the helix, and the Tyr(8) side chain faces the opposite direction from the hydrophobic side. Computational docking prediction has shown that the hydrophobic side of pep-10L sticks in the hydrophobic groove on the GPVI surface, which corresponds to the putative collagen-related peptide binding groove. These data could enable the structure-guided development of a small molecule GPVI antagonist.

DOI： 10.1074/jbc.M808563200

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DOI： 10.1016/j.pnmrs.2008.07.001

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DOI： 10.1007/s10858-008-9276-9

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DOI： 10.1007/s12104-007-9034-z

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DOI： 10.1007/s12104-007-9033-0

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DOI： 10.1074/jbc.M608264200

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DOI： 10.1016/j.jmb.2006.12.037

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DOI： 10.1007/s10858-006-9108-8

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Language：Japanese   Publisher：The Spectroscopical Society of Japan  

Nuclear spin relaxation is one of the fundamental aspects of Nuclear Magnetic Resonance (NMR). Transverse relaxation rate governs the linewidth of the resonance, and longitudinal relaxton rate affects the determination of the recycle delay for each acquisition. On the other hand, cross-relaxation causes the nuclear Overhauser effect (NOE) which gives us distance information used for the 3D structure determination of molecules. Recently, crosscorrelated relaxation has been effectively utilized to develop novel NMR mesaurements. In this review, semi-classical treatment of relaxation theory is briefly intuoduced, and by utilizing the derived double commutator operation, various aspects of nuclear spin relaxation are investigated in terms of molecular motions.

DOI： 10.5111/bunkou.55.205

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Other Link： https://jlc.jst.go.jp/DN/JALC/00281621765?from=CiNii

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW&gt
50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220-223]. In the amide p roton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of 1H2O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach. © Springer 2006.

DOI： 10.1007/s10858-006-0008-8

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DOI： 10.1007/s10858-005-5054-0

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DOI： 10.1007/s10858-005-3870-x

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society  

Membrane-peptide interactions are involved in many crucial biological and pharmacological activities. To clarify the interaction mode of membrane-peptide complexes, it is important to analyze both the dynamic properties and the contact residues of the membrane-bound peptide. In this study, we investigated the dynamic properties of a peptide bound to a lipid bilayer, using relaxation and amide-water exchange analyses, and directly determined the membrane-peptide interface, using the cross-saturation method. For the models of a lipid bilayer and a peptide, isotropic bicelles and mastoparan were used, respectively. The results indicate that mastoparan had a heterogeneous distribution of motion over various timescales and interacted with the lipid bilayer by using its hydrophobic side
the molecule was located within the lipid bilayer rather than on the surface, as thought previously. This study shows that the cross-saturation method is useful for determining the interface of not only protein-protein but also membrane-peptide complexes. © 2005 by the Biophysical Society.

DOI： 10.1529/biophysj.105.066910

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The action mechanism of sapecin, an antibacterial peptide with membrane permeabilization activity, was investigated. The dose dependence of the membrane permeabilization caused by sapecin was sigmoidal, suggesting that sapecin oligomerization leads to the membrane permeabilization. Solution nuclear magnetic resonance analysis of the sapecin-phospholipid vesicle complex revealed the surface buried in the membrane and oligomerization surface on the sapecin molecule. The membrane-buried surface of sapecin was determined by observing the transferred cross-saturation phenomena from the alkyl chains of the phospholipid vesicle to the amide protons of sapecin. The membraneburied surface contains basic and highly exposed hydrophobic residues, which are suitable for interacting with the acidic bacterial membrane. The oligomerization surface was also identified by comparisons between the results from hydrogen-deuterium exchange experiments and transferred cross-saturation experiments. On the basis of the results from the NMR experiments we built a putative model of sapecin oligomers, which provides insights into the membrane permeabilization caused by insect defensins.

DOI： 10.1074/jbc.M307815200

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DOI： 10.1042/BJ20031192

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DOI： 10.1038/nsb876

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S21_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cell Press  

We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K + channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K+ channel. Based on properties of the AgTx2 binding site with the KcsA K+ channel, we present a surface motif that is observed in pore-blocking toxins affecting the K+ channel. Furthermore, we also explain the structural basis of the specificity of the K+ channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.

DOI： 10.1016/j.str.2003.10.003

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DOI： 10.1016/s0022-2836(02)00595-8

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DOI： 10.1016/S0022-2836(02)00018-9

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Language：English   Publisher：2  

Previously, we purified a serine protease with a molecular mass of 26 kDa that exhibits potent antibacterial activity from a pupal extract of <i>Sarcophaga peregrina</i> (flesh fly). We divided this protease into 12 peptides and examined their antibacterial activity. A peptide corresponding to residues 155 to 174 (peptide 9) was found to exhibit antibacterial activity comparable to that of the 26-kDa protease. When <i>Escherichia coli</i> was treated with peptide 9, the permeability of both the outer and inner membranes increased, and substrates for β-lactamase and β-galactosidase entered the cells, but β-galactosidase did not leak out of the cells under these conditions. It was suggested that residues 6 to 18 of peptide 9 form an amphiphilic α-helix under hydrophobic conditions with an N-terminal basic loop and then interact with acidic phospholipids in the bacterial membranes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A 26-mer peptide with the sequence of the pore forming region (residues 427-452) of the Shaker K+ channel (H5 region) was chemically synthesized. Analyses by CD and two-dimensional H-1 NMR spectroscopy were used to investigate the structure of the peptide bound to SDS micelles in solution, which are commonly used in biophysical studies. The tertiary structure of the peptide as a monomer was composed of an a-helix (431-438), a turn (439-442), and random coils (427-430, 443-452), and was very similar to that of the pore forming region of the native K+ channel from Streptomyces lividans determined by X-ray analysis [1]. This result suggests that even an isolated peptide forms a native-like conformation for residues from 431 to 442, depending on its intrinsic amino acid sequence and the surrounding environment. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/s0167-4838(00)00273-9

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K+ channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K+ channel inhibitors. Unlike most other K+ channel-blocking toxins, HaTx1 adopts an 'inhibitor cystine knot' motif and is composed of two β-strands, strand I for residues 19-21 and strand II for residues 28-30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca2+ and Na+ channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage-gated ion channels. (C) 2000 Academic Press.

DOI： 10.1006/jmbi.2000.3609

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Identification of the interfaces of large (M(r) &gt
50,000) protein- protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.

DOI： 10.1038/73331

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 Å for the crystal at pH 5.25 and at 1.55 Å for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of IIe95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro100H-Trp100aH-Phe100bH-Ala101H-Tyr102H. NMR pH titration experiments revealed the pKa values of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (pK(a) = 6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.

DOI： 10.1006/jmbi.1999.2931

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In the hydrolytic reaction catalyzed by an endoglucanase from a Bacillus strain (endoglucanase K), 2 of 12 Trp residues, Trp174 and Trp243, are responsible for binding of the substrate and/or for the catalysis (Kawaminami, S., Ozaki, K., Sumitomo, N., Hayashi, Y., Ito, S., Shimada, I., and Arata, Y. (1994) J. Biol. Chem. 269, 28752-28756). Here we report results of a stable isotope-aided NMR analysis of the active site of endoglucanase K, using Trp174 and Trp243 as structural probes. Hydrogen-deuterium exchange experiments performed for the NH protons of main and side chains of Trp residues revealed that Trp174 and Trp243 are located in the hydrophilic and hydrophobic microenvironments in the active site, respectively. We also carried out pH titration experiments for indole C2 proton resonances of Trp residues and measured the pH dependence of specific activities for wild-type endoglucanase K and its mutants in which Glu or Asp residues are replaced with their respective amide forms. On the basis of the results obtained from the present study, we conclude that (a) Glu130 and Asp191, which are in spatial proximity to Trp174 and Trp243 in the active site, play a crucial role in the enzymatic activity
(b) Glu130 and Asp191 interact with each other in the active site, leading to an increase in the pK(a) values to 5.5 for both amino acid residues
and (c) the pK(a) values of Glu130 and Asp191 would lead to an unusually narrow pH- activity profile of the endoglucanase K.

DOI： 10.1074/jbc.274.28.19823

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DOI： 10.1021/ja982153g

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The solution structure of the B domain of staphylococcal protein A (FB) complexed with the Fc fragment of immunoglobulin G (IgG) is reported. A previous NMR analysis has shown that in solution FB is composed of a bundle of three α-helices, helix I, helix II, and helix III [Gouda, H., Torigoe, H., Saito, A., Sato, M., Arata, Y., and Shimada, I. (1992) Biochemistry 31, 9665-9672]. In contrast, the crystal structure of FB in the FB-Fc complex lacks helix III. Uniformly 15N- and 15N/13C-labeled FB were prepared, and the backbone 13C resonances were assigned. The spectral data obtained in the present study indicated that in solution all three helices including helix III are preserved in the FB-Fc complex. The mode of interaction of FB with the Fc fragment was discussed on the basis of the combined data of hydrogen-deuterium exchange experiments and 1H-15N correlation spectroscopy. It was concluded that a contiguous surface shaped by F14, Y15, E16, L18, and H19 in helix I, and N29, Q33, L35, and K36 in helix II is responsible for the binding.

DOI： 10.1021/bi970923f

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In order to investigate the role of the constant domainson the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalphy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains. © 1995 Federation of European Biochemical Societies.

DOI： 10.1016/0014-5793(95)00113-N

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DOI： 10.1016/s0076-6879(94)39017-7

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Language：English   Publishing type：Research paper (scientific journal)  

An isotope-edited proton nuclear magnetic resonance study is reported of Fv, which is the smallest antigen recognition unit composed of VH and VL domains. Fv has been obtained by clostripain digestion of a short-chain anti-dansyl mouse IgG2a monoclonal antibody [Igarashi, T., Sato, M., Katsube, Y., Takio, K., Tanaka, T., Nakanishi, M., &amp
Arata, Y. (1990) Biochemistry 29, 5727-5733]. A variety of stable-isotope-labeled anti-dansyl Fv analogues have been prepared. The aromatic proton resonances for all Tyr residues of the Fv fragment have been assigned in the absence and presence of t-dansyl-L-lysine by means of isotope-edited homonuclear and heteronuclear two-dimensional NMR experiments. On the basis of the established assignments, it has been concluded that the dansyl ring is bound through Tyr-96H and Tyr-104H to both ends of H3, the third hypervariable region of the heavy chain. We also suggest that the antigen binding results in the formation of a hydrophobic core comprising the dansyl ring and the aromatic rings of Tyr-96H and Tyr-104H. © 1992, American Chemical Society. All rights reserved.

DOI： 10.1021/bi00159a007

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The Fv fragment, which is a smallest antigen-binding unit of immunoglobulin, has been used for a 1H-15N shift correlation NMR study of the dynamical structure of the antibody combining site. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain. We have previously reported that of the six hypervariable regions, three each from the heavy chain (H1, H2, and H3) and the light chain (L1, L2, and L3), H3 is primarily responsible for the antigen binding in the anti-dansyl Fv fragment. The backbone amide nitrogens of all non-proline amino acid residues in H3 have been multiply labeled with 15N. [15N] T2relaxation times and hydrogen-deuterium exchange rates of the amide groups of the main chain were measured in the absence and presence of ϵ-dansyl-L-lysine (DNS-Lys). It has been shown that (1) in the absence of DNS-Lys H3 displays a significant degree of internal motion and (2) antigen binding induces a significant change in the dynamical structure of H3. © 1992, American Chemical Society. All rights reserved.

DOI： 10.1021/bi00124a005

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A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain [Takahashi, H., Igarashi, T., Shimada, I., &amp
Arata, Y. (1991) Biochemistry 30, 2840-2847]. A variety of Fv analogues labeled with 2H in the aromatic rings and with l3C and/or 15N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv in the absence and presence of ϵ-dansyl-L-lysine (DNS-Lys). It has been shown that 1H-15N shift correlation spectra of Fv sensitively reflect the antigen binding and can be used along with 1H and 13C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been followed in the absence and presence of DNS-Lys by using the 1H-15N shift correlation spectra. Use of the β-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed. © 1991, American Chemical Society. All rights reserved.

DOI： 10.1021/bi00240a034

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The Fv fragment, a univalent antigen-binding unit with a molecular weight of 25 000, has successfully been prepared in high yield by limited proteolysis with clostripain of a short-chain mouse IgG2a anti-dansyl monoclonal antibody in which the entire CH1 domain is deleted [Igarashi, T., Sato, M., Takio, K., Tanaka, T., Nakanishi, M., &amp
Arata, Y. (1990) Biochemistry 29, 5727–5733]. The Fv fragment obtained is stable at room temperature and retains its full antigen-binding capability. It has been shown that selective deuterium labeling of the Fv fragment, which is half the size of the Fab fragment, provides Ή NMR spectral data at a sufficient resolution for a detailed structural analysis of the antigen-combining site. NOESY spectra of an Fv analogue, in which all aromatic protons except for His C2′-H and Tyr C3′,5′-H had been deuterated, were measured in the presence of varying amounts of dansyl-L-lysine. On the basis of the NOESY data obtained, it was possible to assign all the ring proton resonances for the dansyl group that is bound to the Fv fragment. It was also possible to obtain information about His and Tyr residues of the Fv fragment in the absence and presence of the antigen. On the basis of the NMR data obtained, we have shown that at least two Tyr residues along with one of the amide groups are directly involved in antigen binding. The mode of interaction of the dansyl ring with these residues in the Fv fragment has briefly been discussed. © 1991, American Chemical Society. All rights reserved.

DOI： 10.1021/bi00225a016

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Language：Japanese   Publisher：The Society of Polymer Science, Japan  

DOI： 10.1295/kobunshi.40.628

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Other Link： https://jlc.jst.go.jp/DN/JALC/00077880512?from=CiNii

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2017.11.713

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：Japanese Proteomics Society (Japan Human Proteome Organisation)  

DOI： 10.14889/jhupo.2003.0.39.0

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.40.S175_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.40.S22_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S131_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S104_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

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Language：English   Publishing type：Book review, literature introduction, etc.  

A comparative NMR structural study of anti-dansyl Fv and Fab fragments is reported. Both of these antigen binding fragments have been prepared using antibodies that originate from the identical anti-dansyl switch variant cell lines. It has been confirmed that the Fv and Fab fragments possess the identical binding property. The antigen binding fragment analogs labeled with 15N of the main chain amide group of the aromatic residues (His, Phe, Trp, and Tyr) were used. The chemical shift and hydrogen-deuterium exchange rate of the amide protons are compared for the Fv and Fab fragments. On the basis of the NMR data obtained, we have concluded that (1) the structural change induced in the VH domain upon antigen binding significantly affects the dynamical structure of the VL domain and (2) the existence of the constant regions affects the fluctuation of the VL domain, increasing the thermal stability of the variable region. © 1994 Academic Press Limited.

DOI： 10.1006/jmbi.1994.1675

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Language：Japanese   Publisher：化学同人  

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Language：Japanese   Publisher：化学同人  

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Other Link： http://search.jamas.or.jp/link/ui/1990117101