記述言語：英語   掲載種別：研究論文（学術雑誌）  

Glioma amplified sequence 41 (GAS41), which has the Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain that recognizes lysine acetylation (Kac), regulates gene expression as a subunit of the SRCAP (SNF2-related CREBBP activator protein) complex that deposits histone H2A.Z at promoters in eukaryotes. The YEATS domains of the proteins AF9 and ENL recognize Kac by hydrogen bonding the aromatic cage to arginine situated just before K9ac or K27ac in the N-terminal tail of histone H3. Curiously, the YEATS domain of GAS41 binds most preferentially to the sequence that contains K14ac of H3 (H3K14ac) but lacks the corresponding arginine. Here, we biochemically and structurally elucidated the molecular mechanism by which GAS41 recognizes H3K14ac. First, stable binding of the GAS41 YEATS domain to H3K14ac required the N terminus of H3 (H3NT). Second, we revealed a pocket in the GAS41 YEATS domain responsible for the H3NT binding by crystallographic and NMR analyses. This pocket is away from the aromatic cage that recognizes Kac and is unique to GAS41 among the YEATS family. Finally, we showed that E109 of GAS41, a residue essential for the formation of the H3NT-binding pocket, was crucial for chromatin occupancy of H2A.Z and GAS41 at H2A.Z-enriched promoter regions. These data suggest that binding of GAS41 to H3NT via its YEATS domain is essential for its intracellular function.

DOI： 10.1073/pnas.2304103120

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The relaxation dispersion (rd) of nuclear magnetic resonance provides thermodynamic and kinetic information regarding molecules for which the conformations are exchanging in equilibrium. Experiments have often been implemented with Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences for heteronuclear S-spin in SI and SI3 spin systems. One of the most common CPMG sequences contains a sequence called a P-element in the middle to average the different relaxation rates of anti-phase and in-phase coherences; however, its drawback is that the artifacts that can be compensated for are only those in one of the two S-spin doublet magnetization components, for example, SIα or SIβ in an SI spin system. Thus, when the CPMG sequence is followed by a normal heteronuclear single-quantum correlation (HSQC) sequence, the detected signal will also include the other component with accumulated artifacts. To overcome this issue, we developed a new pulse sequence (AFTAC) that can suppress artifacts in both the magnetization components. Its effectiveness was demonstrated by simulations and actual measurements targeting the methyl groups of dimethylsulfoxide and N, N-dimethyltrichloroacetamide. The results demonstrated that the AFTAC sequence sufficiently suppressed the artifacts, despite being followed by an HSQC sequence that detects both components. AFTAC is particularly suitable for the rd measurements of small molecules, which are usually not deuterated and are not subject to transverse relaxation optimized spectroscopy (TROSY) sequences. AFTAC does not require 1H continuous wave irradiation for I-spin decoupling, which is necessary for certain CPMG methods that maintain S-spin in-phase coherence during the CPMG period (Tcpmg). Therefore, AFTAC places less burden on the probe, even with a long Tcpmg. Furthermore, the AFTAC method achieves a similar artifact suppression quality not only in SI but also in SI2 and SI3 spin systems.

DOI： 10.1016/j.jmr.2023.107489

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

DOI： 10.1093/nar/gkac1082

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記述言語：英語  

DOI： 10.1021/jacs.2c03580

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier {BV}  

DOI： 10.1074/jbc.RA120.016271

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

13C-direct detection NMR has several advantages compared to proton detection, including a tendency to relax slower and wider chemical shift range. However, the sensitivity of 13C-direct detection is much lower than that of proton detection because of its lower gyromagnetic ratio. In addition, a virtual decoupling procedure is often performed to remove peak splitting in the 13C-direct detection axis, which further reduces the sensitivity to 1/√2. In this study, to enhance the sensitivity of 13C-direct detection experiments, we developed a HCACO-type new pulse sequence in which anti-phase (AP) and in-phase (IP) signals are acquired sequentially in a single scan. The developed experiment was tested on an amino acid (valine) and two proteins (streptococcal protein G B1 domain (GB1) and α-synuclein). The AP and IP spectra were successfully obtained in all cases. Using these spectra, IPAP virtual decoupling was performed, and peak splitting was successfully removed. The sensitivity of the experiment was increased by 1.43, 1.26 and 1.26 times for valine, GB1 and α-synuclein, respectively, compared to the conventional HCACO experiment. In addition, we developed another HCACO-type pulse sequence, where AP and IP signals are simultaneously acquired in a single FID. The sensitivity of the experiment was increased by 1.40 and 1.35 times for valine and GB1, respectively. These methods are potentially applicable to other 13C-direct detection experiments that measure one-bond correlations and will further extend the utility of the 13C-direct detection method, especially for structural analyses of intrinsically disordered proteins.

DOI： 10.1016/j.jmr.2020.106878

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac[Formula: see text](2-3)Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc) and its precursor, asialo-GM1 (Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the [Formula: see text]-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

DOI： 10.1038/s41598-020-78926-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media {LLC}  

DOI： 10.1007/s00216-020-02649-x

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その他リンク： http://link.springer.com/article/10.1007/s00216-020-02649-x/fulltext.html

掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier {BV}  

DOI： 10.1074/jbc.RA119.008735

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1126/science.aau3613

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掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/molecules23102465

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Chitin-binding domain of chitinase A1 (ChBDChiA1 ) is characteristic because it binds only to insoluble crystalline chitin. While binding sites of major carbohydrate-binding modules carry multiple aromatic rings aligned on a surface, lethal mutations for ChBDChiA1 were reported only at W687, a location completely different from the site mentioned above, in spite of their similar main-chain folds. Here, the structural mechanism underlying its crystalline chitin binding was uncovered by solid-state NMR. Based on 13 C- and 15 N-signal assignment of microcrystalline ChBDChiA1 , the chemical shift perturbation on chitin binding was carefully examined. The perturbation was greatest at W687 and nonaromatic residues surrounding it, revealing their direct involvement in chitin binding. These residues and Q679 should provide a novel chitin-binding platform parallel to the W687 ring.

DOI： 10.1002/1873-3468.13226

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掲載種別：研究論文（学術雑誌）   出版者・発行元：{MDPI} {AG}  

DOI： 10.3390/magnetochemistry4030033

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Palgrave Macmillan Ltd.  

Photosystem I (PSI), a large protein complex located in the thylakoid membrane, mediates the final step in light-driven electron transfer to the stromal electron carrier protein ferredoxin (Fd). Here, we report the first structural description of the PSI-Fd complex from Thermosynechococcus elongatus. The trimeric PSI complex binds three Fds in a non-equivalent manner. While each is recognized by a PSI protomer in a similar orientation, the distances between Fds and the PSI redox centres differ. Fd binding thus entails loss of the exact three-fold symmetry of the PSI's soluble subunits, inducing structural perturbations which are transferred to the lumen through PsaF. Affinity chromatography and nuclear magnetic resonance analyses of PSI-Fd complexes support the existence of two different Fd-binding states, with one Fd being more tightly bound than the others. We propose a dynamic structural basis for productive complex formation, which supports fast electron transfer between PSI and Fd.

DOI： 10.1038/s41477-018-0130-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Ferredoxins (FDX) and the FDX:NADP(+) oxidoreductase (FNR) represent a key junction of electron transport downstream of photosystem I (PSI). Dynamic recruitment of FNR to the thylakoid membrane has been considered as a potential mechanism to define the fate of photosynthetically derived electrons. In this study, we investigated the functional importance of the association of FNR with the photosynthetic apparatus in Chlamydomonas reinhardtii. In vitro assays based on NADP(+) photoreduction measurements as well as NMR chemical shift perturbation analyses showed that FNR preferentially interacts with FDX1 compared to FDX2. Notably, binding of FNR to a PSI supercomplex further enhanced this preference for FDX1 over FDX2, suggesting that FNR is potentially capable of channelling electrons towards distinct routes. NADP(+) photoreduction assays and immunoblotting revealed that the association of FNR with the thylakoid membrane including the PSI supercomplex is impaired in the absence of Proton Gradient Regulation 5 (PGR5) and/or Proton Gradient Regulation 5-Like photosynthetic phenotype 1 (PGRL1), implying that both proteins, directly or indirectly, contribute to the recruitment of FNR to the thylakoid membrane. As assessed via in vivo absorption spectroscopy and immunoblotting, PSI was the primary target of photodamage in response to high-light stress in the absence of PGR5 and/or PGRL1. Anoxia preserved the activity of PSI, pointing to enhanced electron donation to O-2 as the source of the observed PSI inactivation and degradation. These findings establish another perspective on PGR5/PGRL1 knockout-related phenotypes and potentially interconnect FNR with the regulation of photosynthetic electron transport and PSI photoprotection in C. reinhardtii.

DOI： 10.1007/s11120-017-0408-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PORTLAND PRESS LTD  

Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum similar to 40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100 mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay. Furthermore, Fd: SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on the addition of SiR to N-15-labeled Fd at the various NaCl concentrations also demonstrated that a combination of electrostatic and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd: SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions.

DOI： 10.1042/bcj20160658

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbabio.2016.03.023

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier Inc.  

Here, we have compiled a nuclear magnetic resonance (NMR)-derived set of nuclear Overhauser enhancement (NOE) distance and dihedral angle restraints that allow for the calculation of the structure of the NDH-1 complex subunit CupS from Thermosynechococcus elongatus in solution. These restraints to calculate the structure in solution of CupS have been deposited to the Protein Data Bank (www.rcsb.org) under PDB-ID accession number 2MXA. This is the first experimental data set published to compute the three-dimensional structure of CupS. This structure is presented in the research article "Solution structure of the NDH-1 complex subunit CupS from Thermosynechococcus elongatus" published by Korste et al. in Biochim. Biophys. Acta 1847(2015)1212-1219 [1]. The cyanobacterial multi-subunit membrane protein complex NDH-1 structurally and functionally relates to Complex I of eubacteria and mitochondria. The NDH-1 complex is mechanistically involved in respiration and cyclic electron transfer around photosystem I (PSI) as well as in a unique mechanism for inorganic carbon concentration.

DOI： 10.1016/j.dib.2015.12.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

The conformational fluctuation in the minimum DNA-binding domain of cMyb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30-70 degrees C, with a transition temperature of approximately 50 degrees C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both 1 H one-dimensional and N-15/H-1 two-dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild-type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity- filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild-type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37 degrees C and is likely beneficial for its biological function: DNA-binding. This result is in agreement with the concept of an excited-state conformer that exists in equilibrium with the dominant ground-state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide-type c-Myb DNA-binding domain to produce an appropriate fraction of the locally fluctuating state at 37 degrees C, which is more amenable to DNA-binding.

DOI： 10.1111/febs.13508

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Å resolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

DOI： 10.1021/acs.biochem.5b00601

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an indirect/reflected detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a chimeric membrane protein-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the prediction tool POODLE and 35 expression vectors were constructed. Subsequently, we obtained N-15-labeled chimeric PH0471 proteins with 25 IDPs as linkers. The NMR spectra allowed us to classify IDPs into three categories: flexible, moderately flexible, and inflexible. The inflexible IDPs contain membrane-associating or aggregation-prone sequences. This is the first attempt to use an indirect/reflected NMR method to evaluate IDPs and can verify the predictions derived from our computational tools.

DOI： 10.3390/ijms160715743

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbabio.2015.05.023

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The cyanobacterial multi-subunit membrane protein complex NDH-1 is both structurally and functionally related to Complex I of eubacteria and mitochondria. In addition to functions in respiration and cyclic electron transfer around photosystem I (PSI), the cyanobacterial NDH-1 complex is involved in a unique mechanism for inorganic carbon concentration. Although the crystal structures of the similar respiratory Complex I from Therms thermophilus and Escherichia coli are known, atomic structural information is not available for the cyanobacterial NDH-1 complex yet. In particular, the structures of those subunits that are not homologous to Complex I will help to understand their distinct functions. The 15.7 kDa protein CupS is a small soluble subunit of the complex variant NDH-1MS, which is thought to play a role in CO2 conversion.
Here, we present the NMR structure of CupS from Thermosynechococcus elongatus, which is the very first structure of a specific cyanobacterial NDH-1 complex subunit. CUPS shares a structural similarity with members of the Fasciclin protein superfamily. The structural comparison to Fasciclin type proteins based on known NMR structures and protein sequences of human TGFBIp, MPB70 from Mycobacterium bovis, and Fdp from Rhodobacter sphaeroides, together with a virtual docking model of CupS and NdhF3, provide first insight into the specific binding of CUPS to the NDH-1MS complex at atomic resolution. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2015.05.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Rap1B is a small GTPase involved in the regulation of numerous cellular processes including synaptic plasticity, one of the bases of memory. Like other members of the Ras family, the active GTP-bound form of Rap1B can bind to a large number of effector proteins and so transmit signals to downstream components of the signaling pathways. The structure of Rap1B bound only to a nucleotide has yet to be solved, but might help reveal an inactive conformation that can be stabilized by a small molecule drug. Unlike other Ras family proteins such as H-Ras and Rap2A, Rap1B crystallizes in an intermediate state when bound to a non-hydrolyzable GTP analog. Comparison with H-Ras and Rap2A reveals conservative mutations relative to Rap1B, distant from the bound nucleotide, which control how readily the protein may adopt the fully activated form in the presence of GTP. High resolution crystallographic structures of mutant proteins show how these changes may influence the hydrogen bonding patterns of the key switch residues. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.bbrc.2015.04.103

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T (1) relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T (1) values across the entire sequence of the intrinsically disordered protein alpha-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in 'proton-less' NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from alpha(1)-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.

DOI： 10.1007/s10858-015-9925-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. An IgG1 molecule, which is now mainly used for antibody preparation, consists of a total of 12 immunoglobulin domains. Each domain has one disulfide bond. The C(H)3 domain is the C-terminal domain of the heavy chain of IgG1. The disulfide bonds of some of the C(H)3 domains are known to be reduced in recombinant human monoclonal antibodies. The lack of intramolecular disulfide bonds may decrease the stability and increase the aggregation propensity of an antibody molecule. To investigate the effects of a reduced disulfide bond in the C(H)3 domain on conformational stability and aggregation propensity, we performed several physicochemical measurements including circular dichroism, differential scanning calorimetty (DSC), and 2D NMR. DSC measurements showed that both the stability and reversibility of the reduced form were lower than those of the oxidized form. In addition, detailed analyses of the thermal denaturation data revealed that, although a dominant fraction of the reduced form retained a stable dimeric structure, some fractions assumed a less-specifically associated oligomeric state between monomers. The results of the present study revealed the characteristic aggregation properties of antibody molecules. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2015.02.020

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

ループに富む蛋白質のNMR構造決定法の開発

DOI： 10.1007/s10858-014-9882-7

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：SPRINGER  

Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide by using six electrons transported from ferredoxin (Fd) for eventual sulfur assimilation. As efficient electron flows are ensured by forming a productive Fd:SiR complex, detailed characterization of a Fd:SiR complex in solution is of particular importance. Here, we show that acidic residues of Fd play essential roles in forming an electron transfer complex with SiR by using attractive electrostatic interactions with putative basic residues of SiR. The thermodynamic approach using calorimetry revealed a favorable electrostatic contribution to form the Fd:SiR complex at the molecular level. Solution-state nuclear magnetic resonance (NMR) spectroscopy on N-15-labeled Fd in the presence of SiR showed large perturbations in NMR signals of acidic residues of Fd. The addition of NaCl diminished overall perturbations of NMR signals of SiR-bound Fd which resulted from the decrease in interprotein affinity. However, acidic residues at both termini still showed relatively large peak perturbation. These results at the residue level suggested that intermolecular interactions between Fd and SiR are electrostatic in nature and the electrostatic interaction is a dominant contributor to form the Fd:SiR complex. We suggest that a combination of calorimetry and NMR is a powerful approach to investigating protein-protein interactions.

DOI： 10.1007/978-3-319-20137-5_17

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

NMR structure determination of soluble proteins depends in large part on distance restraints derived from NOE. In this study, we examined the impact of paramagnetic relaxation enhancement (PRE)-derived distance restraints on protein structure determination. A high-resolution structure of the loop-rich soluble protein Sin1 could not be determined by conventional NOE-based procedures due to an insufficient number of NOE restraints. By using the 867 PRE-derived distance restraints obtained from the NOE-based structure determination procedure, a high-resolution structure of Sin1 could be successfully determined. The convergence and accuracy of the determined structure were improved by increasing the number of PRE-derived distance restraints. This study demonstrates that PRE-derived distance restraints are useful in the determination of a high-resolution structure of a soluble protein when the number of NOE constraints is insufficient.

DOI： 10.1007/s10858-014-9882-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

The cyanobacterial NDH-1 complex is involved in respiratory as well as in cyclic electron transfer around photosystem I. Here, we report both backbone and side chain chemical shift assignments of CupS, a small subunit of the multisubunit membrane protein complex NDH-1 from Thermosynechococcus elongatus. The construct contains 159 amino acids including a Strep-tag and two additional amino acids.

DOI： 10.1007/s12104-014-9567-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.

DOI： 10.1073/pnas.1322602111

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

In order to develop a structure-based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on N,N-diacetylchitobiose deacetylases (Dacs) from Pyrococcus horikoshii (Ph-Dac) and Pyrococcusfuriosus (Pf-Dac). Neither Ph-Dac nor Pf-Dac was expressed in the soluble fraction of Escherichiacoli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E.coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were active and thermostable up to 85 degrees C. The crystal structures of Ph-Dac and Pf-Dac were determined at resolutions of 2.0 angstrom and 1.54 angstrom, respectively. The Pyrococcus Dac forms a hexamer composed of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph-Dac, catalytic Zn situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N-acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the N epsilon atom of His264 (from the adjacent polypeptide in the Ph-Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. Mutation analysis also indicated that His264 was essential to the catalysis. These factors give the archaeal Dacs an unprecedented active site architecture a Zn-dependent deacetylases. Database Structural data are available in the Protein Data Bank database under accession numbers 3WL3, 3WL4, and 3WE7. Structured digital abstract Ph-DacandPh-Dacbindbyx-ray crystallography(View interaction) Pf-DacandPf-Dacbindbyx-ray crystallography(View interaction)

DOI： 10.1111/febs.12805

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 degrees C and was insensitive to the number of heating (90 degrees C)-cooling (20 degrees C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

DOI： 10.1074/jbc.m113.534222

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Intrinsically disordered domains have been reported to play important roles in signal transduction networks by introducing cooperativity into protein-protein interactions. Unlike intrinsically disordered domains that become ordered upon binding, the EF-SAM domain in the stromal interaction molecule (STIM) 1 is distinct in that it is ordered in the monomeric state and partially unfolded in its oligomeric state, with the population of the two states depending on the local Ca2+ concentration. The oligonnerization of STIM1, which triggers extracellular Ca2+ influx, exhibits cooperativity with respect to the local endoplasmic reticulum Ca2+ concentration. Although the physiological importance of the oligomerization reaction is well established, the mechanism of the observed cooperativity is not known. Here, we examine the response of the STIM1 EF-SAM domain to changes in Ca2+ concentration using mathematical modeling based on in vitro experiments. We find that the EF-SAM domain partially unfolds and dimerizes cooperatively with respect to Ca2+ concentration, with Hill coefficients and half-maximal activation concentrations very close to the values observed in vivo for STIM1 redistribution and extracellular Ca2+ influx. Our mathematical model of the dimerization reaction agrees quantitatively with our analytical ultracentrifugation-based measurements and previously published free energies of unfolding. A simple interpretation of these results is that Ca2+ loss effectively acts as a denaturant, enabling cooperative dimerization and robust signal transduction. We present a structural model of the Ca2+-unbound EF-SAM domain that is consistent with a wide range of evidence, including resistance to proteolytic cleavage of the putative dimerization portion. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

DOI： 10.1016/j.jmb.2014.03.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

SAPK-interacting protein 1 (Sin1) is an important component of the target of rapamycin (TOR) complex 2 (TORC2). TOR is a serine/threonine-specific protein kinase and forms functionally distinct protein complexes referred to as TORC1 and TORC2. TORC2, conserved from yeast to humans, phosphorylates AGC-family protein kinases and has many cellular functions including the regulation of actin cytoskeleton. The Sin1 subunit of TORC2 is required for the binding of TORC2 to substrates, and the conserved region in the middle (CRIM) domain of Sin1 is important in the substrate recognition of TORC2. Here, we report on the H-1, C-13 and N-15 resonance assignments of fission yeast Schizosaccharomyces pombe Sin1 (amino acids 247-400) (Sin1(CRIM)), which possesses the CRIM domain. These data contribute toward the structure determination of Sin1(CRIM) and an understanding of the interactions of Sin1(CRIM) with substrates of TORC2.

DOI： 10.1007/s12104-014-9550-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The stability of an SR398/GroES chaperonin complex was examined. As was expected, based on the finding of previous studies, the SR398/GroES complex was extremely stable in the presence of an excess amount of free adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) or adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP). However, the complex was not stable in the absence of nucleotides. These results indicate that ATP gamma S and AMPPNP repeatedly associated to and dissociated from the complex in a non-cooperative manner. This nucleotide exchange did not induce the dissociation of GroES and substrate from SR398, suggesting the importance of the cooperative dissociation of nucleotides from the cis-ring to release GroES and substrate proteins in the GroEL/GroES reaction cycle.

DOI： 10.1093/jb/mvu009

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

There are structural and functional differences in SmtB homologs, metal-responsive transcription factors responsible for sensing of excess heavy metal ions in marine and freshwater cyanobacterial strains. The structure of SmtB from freshwater Synechococcus sp. strain PCC 7942 is elucidated with nuclear magnetic resonance (NMR) and crystallography techniques. But knowledge about the functioning of SmtB homologs from marine species is limited till date. To enable NMR spectroscopic studies for investigating structural and functional aspects, modified protocols with higher yields of isotopically labeled SmtB, from marine species like Synechococcus sp. PCC 7002 are essential. In this study, smtB gene was cloned from genome of Synechococcus sp. PCC 7002 and overexpression protocol for recombinant SmtB was standardized in Escherichia coli containing T7 RNA polymerase/promoter system. Further, the protocol for large-scale production, isotope labeling with N-15, and purification of recombinant SmtB in E. coli BL21(DE3)/pLysS cells was developed. Purified recombinant protein was successfully used for NMR spectroscopy experiments. These results indicate that the overexpression technique now developed is applicable to the structural and functional studies for the proteins being homologous to cyanobacterial SmtB from Synechococcus sp. PCC 7002.

DOI： 10.1007/s10930-013-9525-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

A chitinase, from Pyrococcus furiosus, is a hyperthermophilic glycosidase that effectively hydrolyses both alpha and beta crystalline chitin. This chitinase has unique structural features; it contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBD1 and ChBD2). We have determined the structure of ChBD1, which significantly enhances the activity of the catalytic domains, by nuclear magnetic resonance spectroscopy. The overall structure of ChBD1 had a compact and globular architecture consisting of three anti-parallel beta-strands, similar to those of other proteins classified into carbohydrate-binding module (CBM) family 5. A mutagenesis experiment suggested three solvent-exposed aromatic residues (Tyr112, Trp113 and Tyr123) as the chitin-binding sites. The involvement of Tyr123 or the corresponding aromatic residues in other CBMs, has been demonstrated for the first time. This result indicates that the binding mode may be different from those of other chitin-binding domains in CBM family 5. In addition, the binding affinities of ChBD1 and ChBD2 were quite different, suggesting that the two ChBDs each play a different role in efficiently increasing the activities of AD1 and AD2.

DOI： 10.1093/jb/mvt104

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE INC  

The side-chain conformations of amino acids in the hydrophobic core are important for protein folding and function. A previous NMR study has shown that a mutant protein of transcriptional activator c-Myb,I155L/I181L R3, has multiple conformations and increased fluctuation in comparison with the wild type. To elucidate the quantitative correlation of structural fluctuation with stability and function, we analyzed the thermodynamic effects of I155L and I181L mutations, using R2R3 that encompasses the minimum specific DNA-binding region. Circular dichroism and differential scanning calorimetry measurements showed that the mutation of I155L had little effect on stability, while the I181L mutation significantly destabilized the protein. It is noteworthy that the decreased stability resulting from the I181L mutation was mainly due to decreased enthalpy change, which is partially compensated by decreased entropy change. Isothermal titration calorimetry measurements showed that the specific DNA-binding affinity was decreased owing to the I181L mutation, which was due to decreased binding entropy change. Entropy in the folded state, which corresponds to the DNA-free state, increases due to the I181L mutation because of the increased conformational fluctuation observed in I155L/I181L mutant of R2R3 by CLEANEX-PM NMR analysis, which in turn results in decreased folding entropy and DNA-binding entropy changes. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2013.07.014

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III domains, and a C-terminal chitin-binding domain (ChBD). The ChBD of ChiA1 (ChBD(ChiA1)) belongs to carbohydrate-binding module (CBM) family 12 and specifically binds to insoluble or crystalline chitin. It has been suggested that tryptophan-687 (Trp687) is involved in the chitin-binding activity of this ChBD. Site-directed mutagenesis was used to identify additional amino acid residues required for chitin-binding activity of this domain. Furthermore, a total of 14 amino acid residues in ChBD(ChiA1) were carefully selected, and it was found that mutation of Gln679, which is not well-conserved in CBM family 12, significantly decreased the binding activity to colloidal chitin. A nuclear magnetic resonance study demonstrated that neither the Q679A nor the W687A mutation altered the overall structure of ChBD(ChiA1). Therefore, Gln679 was identified as a new residue that is involved in the chitin-binding activity of ChBD(ChiA1) in addition to Trp687. However, the mechanism of chitin binding by ChBD is still unknown.

DOI： 10.1093/jb/mvt043

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Escherichia coli is a versatile, low-cost, and popular host for expressing recombinant proteins. However, extracting recombinant proteins from E. coli requires cell wall breakage, which is both time- and effort-consuming. Here we report a novel cell breakage method based on our recent finding that VanX, which is a D-Ala-D-Ala dipeptidase encoded in a vancomycin-resistant VanA gene cluster, exhibits a strong cell lysis activity when expressed in isolation in E. coli. In our strategy, we coexpress VanX with the target protein, causing cell autolysis and release of the cellular content into the culture medium. We demonstrated this strategy for two model proteins, a green fluorescent protein variant (GFPuv) and Gaussia luciferase, and optimized the autolysis conditions and coexpression vectors. The fluorescence activity of GFPuv collected from the medium was identical to that of GFPuv purified by conventional methods. Cell breakage by VanX-mediated autolysis is very simple to implement and will efficiently complement traditional methods. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2013.04.007

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.m112.439075

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ferredoxin-NADP(+) reductase (FNR) forms a 1:1 complex with ferredoxin (Fd), and catalyzes the electron transfer between Fd and NADP(+). In our previous study, we prepared a series of site-specifically cross-linked complexes of Fd and FNR, which showed diverse electron transfer properties. Here, we show that X-ray crystal structures of the two different Fd-FNR cross-linked complexes form oligomers by swapping Fd and FNR moieties across the molecules; one complex is a dimer from, and the other is a successive multimeric form. In order to verify whether these oligomeric structures are formed only in crystal, we investigated the possibility of the oligomerization of these complexes in solution. The mean values of the particle size of these cross-linked complexes were shown to increase with the rise of protein concentration at sub-milimolar order, whereas the size of dissociable wild-type Fd:FNR complex was unchanged as analyzed by dynamic light scattering measurement. The oligomerization products were detected by SDS-PAGE after chemical cross-linking of these complexes at the sub-milimolar concentrations. The extent and concentration-dependent profile of the oligomerizaion were differentiated between the two cross-linked complexes. These results show that these Fd-FNR cross-linked complexes exhibit concentration-dependent oligomerization, possibly through swapping of Fd and FNR moieties also in solution. These findings lead to the possibility that some native multi-domain proteins may present similar phenomenon in vivo.

DOI： 10.1016/j.bbrc.2013.04.033

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s10858-012-9675-9

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially N-15-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [H-1, N-15] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) [14]. A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR = 1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.07.083

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In response to an increased level of Zn2+, Synechococcus sp. PCC 7942 expresses SmtA, a metallothionein-like metal-chelating protein, while Synechocystis sp. PCC 6803 expresses ZiaA, a transporter of Zn2+. The gene expression of these proteins is regulated by repressor protein, SmtB and ZiaR, respectively. In spite of contributing to different response systems, both repressor proteins belong to the ArsR family and are highly homologous to each other. To understand the different systems responsible for dealing with excess Zn2+, we examined the cis-elements in the promoter regions of smtA and ziaA, as well as the binding affinities of recombinant SmtB and ZiaR proteins. The operator/promoter region of smtA included two palindromic sequences and that of ziaA included one. Electrophoretic mobility shift assay revealed that SmtB formed four different complexes with the operator/promoter region of smtA, whereas it formed only two different complexes with the corresponding region of ziaA. For ZiaR, the corresponding results were quite the same as those for SmtB. Furthermore, the complex formation between SmtB and operator/promoter regions is inhibited in the presence of Zn2+ at higher concentrations than 16 μM. On the other hand, the corresponding Zn2+ concentration is 128 μM. These results demonstrate that the degrees of protein- DNA complex formation between repressor proteins and the operator/promoter regions of regulated genes depend on the structures of the operator/promoter regions, and the effects of Zn2+ on the dissociation of these complexes are mainly associated with the structures of the repressors. © 2012 TTHE BIOPHYSICAL SOCIETY OF JAPAN.

DOI： 10.2142/biophysics.8.103

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (H2O)-H-2 should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry 50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (H2O)-H-2, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements.

DOI： 10.1021/bi300495p

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jmb.2012.05.034

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

A chitinase from the hyperthermophilic archaeon Pyrococcus furiosus degrades chitin to produce diacetylchitobiose [(GIcNAc)(2)] as the end product. To further investigate the degradation mechanism of (GIcNAc)(2) in Pyrococcus spp., we cloned the gene of PH0499 from Pyrococcus horikoshii, which encodes a protein homologous to the diacetylchitobiose deacetylase of Thermococcus kodakaraensis. The deacetylase (Ph-Dac) was overexpressed as inclusion bodies in Escherichia coli Rosetta (DE3) pLys. The insoluble inclusion body was solubilized and reactivated through a refolding procedure. After several purification steps, 40 mg of soluble, thermostable (up to 80 degrees C) Ph-Dac was obtained from 1 L of culture. The apparent molecular mass of the refolded Ph-Dac was 180 kDa, indicating Ph-Dac to be a homohexamer. The refolded Ph-Dac also exhibited deacetylase activity toward (GIcNAc)(2), and the deacetylation site was revealed to be specific to the nonreducing end residue of (GIcNAc)(2). These expression and purification systems are useful for further characterization of Ph-Dac. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2012.06.002

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Chaperonin GroEL and its partner GroES assist the folding of nascent and stress-damaged proteins in an ATP-dependent manner. Free GroES has a flexible "mobile loop" and binds to GroEL through the residues at the tip of the loop, capping the central cavity of GroEL to provide the substrate polypeptide a cage for secure in-cage folding. Here, we show that restriction of the flexibility of the loop by a disulfide cross-linking between cysteines within the loop results in the inefficient formation of a stable GroEL-polypeptide-GroES ternary complex and inefficient folding. Then, we generated substrate proteins with enhanced binding affinity to GroEL by fusion of one or two SBP (strongly binding peptide for GroEL) sequences and examined the effect of disulfide cross-linking on the assisted folding. The results indicate that the higher the binding affinity of the substrate polypeptide to GroEL, the greater the contribution of the mobile loop flexibility to efficient in-cage folding. It is likely that the flexibility helps GroES capture GroEL's binding sites that are already occupied by the substrate polypeptide with various binding modes. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2012.05.026

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Ferredoxin (Fd), which plays a pivotal role in photosynthesis as an electron carrier, forms a transient complex with various Fd-dependent enzymes, such as nitrite reductase (NiR), to achieve efficient intermolecular electron transfer. We studied the protein-protein interaction of Fd and NiR by NMR spectroscopy and determined three acidic regions of Fd to be major sites for the interaction with NiR, indicating that the complex is stabilized through electrostatic interaction. During this study, we found Fds from higher plant and cyanobacterium, in spite of their high structural similarities including the above acidic regions, differ remarkably in the interaction with cyanobacterial NiR. In activity assay of NiR, K-m value for maize Fd (74.6 mu M) was 9.6 times larger than that for Leptolyngbya boryana Fd (7.8 mu M). The two Fds also showed a similar difference in binding assay to NiR-immobilized resin. Comparative site-specific mutagenesis of two Fds revealed that their discriminative ability for the interaction with NiR is attributed mainly to non-charged residues in the peripheral region of [2Fe-2S] cluster. These non-charged residues are conserved separately between Fds of plant and cyanobacterial origins. Our data highlight that intermolecular force(s) other than electrostatic attraction is(are) also crucial for the molecular interaction between Fd and partner enzyme.

DOI： 10.1093/jb/mvs028

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Differences in the conformation of the pleckstrin homology (PH) domain of switch-associated protein-70 (SWAP-70) in solution and at the lipid bilayer membrane surface were examined using CD, fluorescence and NMR spectroscopy. Intracellular relocalization of SWAP-70 from the cytoplasm to the plasma membrane and then to the nucleus is associated with its cellular functions. The PH domain of SWAP-70 contains a phosphoinositide-binding site and a nuclear localization signal, which localize SWAP-70 to the plasma membrane and nucleus, respectively. CD and fluorescence spectra showed that a significant conformational alteration involving formation of disordered structure occurs when the PH domain binds to d-myo-phosphatidylinositol 3,4,5-trisphosphate or d-myo-phosphatidylinositol 4,5-bisphosphate embedded in lipid bilayer vesicles. NMR spectra indicate that Ala and Trp residues located in the C-terminal alpha-helix of the PH domain undergo conformational alterations to form a disordered structure at the vesicle surface. These conformational alterations were not induced by association with inositol 1,3,4,5-tetrakisphosphate in solution or coexistence of phosphatidylcholine vesicles. Interaction with the plane of the lipid bilayer via association with the phosphoinositides is required for the unfolding of the C-terminal alpha-helix of the PH domain. The unwinding of the C-terminal alpha-helix could regulate the functions of SWAP-70 at the plasma membrane surface.

DOI： 10.1093/jb/mvr146

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 angstrom-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked alpha beta beta alpha core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T-m = 52 degrees C, enhanced the protein thermostability by 36 degrees C (T-m = 88 degrees C), whereas a single mutation (G111S or L137A) decreased the stability by similar to 10 degrees C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 degrees C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

DOI： 10.1074/jbc.m111.321992

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

To obtain insight into the motional features of proteins for enzymatic function, we studied binding reactions between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) using isothermal titration calorimetry and NMR-based magnetic relaxation and hydrogen/deuterium exchange (HDex). Fd-FNR binding was accompanied by endothermic reactions and driven by the entropy gain. Component-wise analysis of the net entropy change revealed that increases in the conformational entropy of the Fd-FNR complex contributed largely to stabilizing the complex. Intriguingly, analyses of magnetic relaxation and HDex rates with X-ray B factor implied that Fd binding led to both structural stiffening and softening of FNR. Enhanced FNR backbone fluctuations suggest favorable contributions to the net conformational entropy. Fd-bound FNR further showed that relatively large-scale motions of the C terminus, a gatekeeper for interactions of NADP(+)(H), were quenched in the closed form, thereby facilitating exit of NADP(+)(H). This can provide a first dynamic structure-based explanation for the negative cooperativity between Fd and NADP(+)(H) via FNR.

DOI： 10.1002/cbic.201100189

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PORTLAND PRESS LTD  

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues I to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys(51), Lys(53), Arg(177) and Arg(179), in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1-211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.

DOI： 10.1042/bj20110241

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Zonula occludens-1 (ZO-1) is a scaffolding molecule critical to the formation of intercellular adhesion structures, such as tight junctions (TJs) and adherens junctions (AJs). ZO-1 contains three PDZ domains followed by a GUK domain and a ZU5 domain. The first PDZ of ZO-1 (ZO-1(PDZ1)) serves as a protein-protein interaction module and interacts with the C-termini of almost all claudins to initiate the formation of a belt-like structure on the lateral membranes, thereby promoting TJ formation. It has been recently reported that approximately 15% of all PDZ domains bind phosphoinositides, and ZO-1(PDZ1) is the one of these. Here we report the N-15, C-13, and H-1 chemical shift assignments of the first PDZ domain of mouse ZO-1. The resonance assignments obtained in this work may contribute in clarifying the interplay between the two binary interactions, ZO-1(PDZ1)-claudins and ZO-1(PDZ1)-phospholipids, and suggesting a novel regulation mechanism underlying the formation and maintenance of cell-cell adhesion machinery downstream of the phospholipid signaling pathways.

DOI： 10.1007/s12104-011-9301-x

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbapap.2011.03.009

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jmb.2010.11.029

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JOHN WILEY & SONS INC  

It is challenging to investigate the structure and dynamics of amyloid fibrils at the residue and atomic resolution because of their high molecular weight and heterogeneous properties Here, we used solution nuclear magnetic resonance (NMR) spectroscopy to characterize the conformation and flexibility of amyloid fibrils of beta(2)-microglobulin (beta 2m), for which direct observation of solution NMR could not be made Ultrasonication led to fragmentation producing a solution of minimum-sized fibrils with a molecular weight of around 6 MDa In (1)H-(15)N heteronuclear single-quantum correlation measurements, five signals, derived from N-terminal residues (i e, Ile1, Gln2, Arg3, Thr4, and Lys6), were newly detected Signal strength decreased with the distance from the N-terminal end Capping experiments with the unlabeled beta 2m monomer indicated that the signals originated from molecules located inside the fibrils Ultrasonication makes the residues with moderate flexibility observable by reducing size of the fibrils Thus, solution NMR measurements of ultrasonicated fibrils will be promising for studying the structure and dynamics of fibrils

DOI： 10.1002/pro.515

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Ferredoxin (Fd) and Fd-NADP(+) reductase (FNR) are redox partners responsible for the conversion between NADP(+) and NADPH in the plastids of photosynthetic organisms. Introduction of specific disulfide bonds between Fd and FNR by engineering cysteines into the two proteins resulted in 13 different Fd-FNR cross-linked complexes displaying a broad range of activity to catalyze the NADPH-dependent cytochrome c reduction. This variability in activity was thought to be mainly due to different levels of intramolecular electron transfer activity between the FNR and Fd domains. Stopped-flow analysis revealed such differences in the rate of electron transfer from the FNR to Fd domains in some of the cross-linked complexes. A group of the cross-linked complexes with high cytochrome c reduction activity comparable to dissociable wild-type Fd/FNR was shown to assume a similar Fd-FNR interaction mode as in the native Fd: FNR complex by analyses of NMR chemical shift perturbation and absorption spectroscopy. However, the intermolecular electron transfer of these cross-linked complexes with two Fd-binding proteins, nitrite reductasc and photosystem I, was largely inhibited, most probably due to steric hindrance by the FNR moiety linked near the redox center of the Fd domain. In contrast, another group of the cross-linked complexes with low cytochrome c reduction activity tends to mediate higher intermolecular electron transfer activity. Therefore, reciprocal relationship of intramolecular and intermolecular electron transfer abilities was conferred by the linkage of Fd and FNR, which may explain the physiological significance of the separate forms of Fd and FNR in chloroplasts.

DOI： 10.1021/bi100855a

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Elucidation of the basis of interactions between biological molecules is essential for the understanding of living systems. Src-homology 3 (SH3) domains play critical roles in interaction networks of proteins by recognizing a proline-rich sequence motif, PxxP. There are, however, several SH3 domains that specifically bind to polypeptide chains without the conventional recognition sequence. The SH3 domain of DDEF1 associates with the SA M P motifs of the adenomatous polyposis colt (A PC) tumor suppressor. The SAMP motifs are indispensable for the normal function of APC in tumor suppression. Here we present the structural basis of the interaction between the DDEF1-SH3 domain and the A PC-SA M P motifs. We determined the solution structures of the DDEF1-SH3 domain both in a free state and in a complex with APC-SAMP. As the affinity of the interaction was not sufficiently high for the determination of the complex structure in solution by conventional methods, we utilized a fusion protein of the DDEF1-SH3 domain and APC-SAMP. The structures revealed that the SAMP motif adopts a class II polyproline type II helix even though it does not contain the PxxP motif and that a characteristically large hydrophobic pocket of the SH3 domain confers high selectivity to the interaction. Furthermore, investigation into the backbone dynamics of the free and bound systems by NMR spin relaxation experiments demonstrated that the DDEF1-SH3 domain exhibits high flexibility at the peptide recognition site in the absence of the ligand and that most residues of the APC-SAMP motif display extensive local motions even in the stable complex.

DOI： 10.1021/bi100563z

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 angstrom resolution. The TDH tetramer forms a central pore with dimensions of 23 angstrom in diameter and similar to 50 angstrom in depth. pi-Cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

DOI： 10.1074/jbc.m109.074526

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.

DOI： 10.1074/jbc.m110.108365

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

Although whole-parasite vaccine strategies for malaria infection have regained attention, their immunological mechanisms of action remain unclear. We find that immunization of mice with a crude blood stage extract of the malaria parasite Plasmodium falciparum elicits parasite antigen-specific immune responses via Toll-like receptor (TLR) 9 and that the malarial heme-detoxification byproduct, hemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect. Malarial and synthetic (s)HZ bound TLR9 directly to induce conformational changes in the receptor. The adjuvant effect of sHZ depended on its method of synthesis and particle size. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. The adjuvant function of sHZ was further validated in a canine antiallergen vaccine model. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development.

DOI： 10.1016/j.chom.2009.12.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER INST PHYSICS  

We have developed a new approach to accurately calculate entropy change based on density functional theory in the energy representation. The entropy change was evaluated using the derived equation and energy distributions computed using molecular simulation and reweighting techniques. This approach was applied to a harmonic oscillator, an alanine dipeptide, and a small protein. We found that the results were accurate compared to conventional approaches, such as the quasiharmonic approximation.

DOI： 10.1063/1.3272029

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC PLANT BIOLOGISTS  

Translocation of nuclear-encoded preproteins across the inner envelope of chloroplasts is catalyzed by the Tictranslocon, consisting of Tic110, Tic40, Tic62, Tic55, Tic32, Tic20, and Tic22. Tic62 was proposed to act as a redox sensor of the complex because of its redox-dependent shuttling between envelope and stroma and its specific interaction with the photosynthetic protein ferredoxin-NADP(H) oxidoreductase (FNR). However, the nature of this close relationship so far remained enigmatic. A putative additional localization of Tic62 at the thylakoids mandated further studies examining how this feature might be involved in the respective redox sensing pathway and the interaction with its partner protein. Therefore, both the association with FNR and the physiological role of the third, thylakoid-bound pool of Tic62 were investigated in detail. Coexpression analysis indicates that Tic62 has similar expression patterns as genes involved in photosynthetic functions and protein turnover. At the thylakoids, Tic62 and FNR form high molecular weight complexes that are not involved in photosynthetic electron transfer but are dynamically regulated by light signals and the stromal pH. Structural analyses reveal that Tic62 binds to FNR in a novel binding mode for flavoproteins, with a major contribution from hydrophobic interactions. Moreover, in absence of Tic62, membrane binding and stability of FNR are drastically reduced. We conclude that Tic62 represents a major FNR interaction partner not only at the envelope and in the stroma, but also at the thylakoids of Arabidopsis thaliana and perhaps all flowering plants. Association with Tic62 stabilizes FNR and is involved in its dynamic and light-dependent membrane tethering.

DOI： 10.1105/tpc.109.069815

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are membrane proteins that are widely conserved from bacteria to mammals. The molecular functions of these proteins have not been established. In mammals, the domain is often found in raft-associated proteins such as flotillin and podocin. We determined the structure of the SPFH domain of PH0470 derived from Pyrococcus horikoshii using NMR. The structure closely resembles that of the SPFH domain of the paralog PH1511, except for two C-terminal helices. The results show that the SPFH domain forms stable dimers, trimers, tetramers, and multimers, although it lacks the coiled-coil region for oligomerization, which is a highly conserved region in this protein family. The oligomers exhibited unusual thermodynamic behavior, as determined by circular dichroism, NMR, gel filtration, chemical cross-linking, and analytical ultracentrifugation. The oligomers were converted into monomers when they were heated once and then cooled. This transition was one-way and irreversible. We propose a mechanism of domain swapping for forming dimers as well as successive oligomers. The results of this study provide what to our knowledge are new insights into the common molecular function of the SPFH domain, which may act as a membrane skeleton through oligomerization by domain swapping.

DOI： 10.1016/j.bpj.2009.07.034

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCI LTD  

Ten C-glycosyl beta(2)- and beta/beta(2)-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble beta(2)-amino acids in which C-glycosylic substituents are attached to the C-2 position of beta-amino acids. Conformational analysis of the C-glycosyl beta(2)-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl beta/beta(2)-peptides are governed by the configuration at the carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl beta(2)-amino acids. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2009.01.018

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Post-translational modification by small ubiquitin-like modifier (SUMO) proteins has been implicated in the regulation of a variety of cellular events. The functions of sumoylation are often mediated by downstream effector proteins harboring SUMO-interacting motifs (SIMs) that are composed of a hydrophobic core and a stretch of acidic residues. MBD1-containing chromatin-associated factor 1 (MCAF1), a transcription repressor, interacts with SUMO-2/3 and SUMO-1, with a preference for SUMO-2/3. We used NMR spectroscopy to solve the solution structure of the SIM of MCAF1 bound to SUMO-3. The hydrophobic core of the SIM forms a parallel beta-sheet pairing with strand beta2 of SUMO-3, whereas its C-terminal acidic stretch seems to mediate electrostatic interactions with a surface area formed by basic residues of SUMO-3. The significance of these electrostatic interactions was shown by mutations of both SUMO-3 and MCAF1. The present structural and biochemical data suggest that the acidic stretch of the SIM of MCAF1 plays an important role in the binding to SUMO-3.

DOI： 10.1074/jbc.m802528200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The adenomatous polyposis coli (APC) tumor suppressor protein is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and in cytoskeletal regulation. The SAMP repeats region of APC, an Axin-binding site, is known to be important for tumor suppression and for the developmental function of APC. We performed a yeast two-hybrid screening using the first SAMP motif-containing region of Xenopus APC as bait and obtained several SAMP binding candidates including DDEF2 (development and differentiation enhancing factor 2), which is an ADP-ribosylation factor (Arf) GTPase-activating protein (GAP (ArfGAP)) involved in the regulation of focal adhesions. In vitro and in cells the Src homology 3 (SH3) domain of DDEF2 and its close homolog, DDEF1, are associated with the SAMP motif of APC competitively with Axin1. Moreover, NMR chemical shift perturbation experiments revealed that the SAMP motif interacts at the same surface of the SH3 domain of DDEF as the known SH3 binding motif, PXXP. When fluorescent protein-tagged APC and DDEF are expressed in Xenopus A6 cells, co-localization at microtubule ends is observed. Overexpression and RNA interference experiments indicate that APC and DDEFs cooperatively regulate the distributions of microtubules and focal adhesions. Our findings reveal that the SAMP motif of APC specifically binds to the SH3 domains of DDEFs, providing new insights into the functions of APC in cell migration.

DOI： 10.1074/jbc.m800420200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both alpha and beta crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded beta-sheets, was composed of a typical beta-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2008.06.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The malaria parasite possesses plant-type ferredoxin (Fd) and ferredoxin-NADP(+) reductase (FNR) in a plastid-derived organelle called the apicoplast. This Fd/FNR redox system, which potentially provides reducing power for essential biosynthetic pathways in the apicoplast, has been proposed as a target for the development of specific new anti-malarial agents. We studied the molecular interaction of Fd and FNR of human malaria parasite (Plasmodium falciparum), which were produced as recombinant proteins in Escherichia coli. NMR chemical shift perturbation analysis mapped the location of the possible FNR interaction sites on the surface of P. falciparum Fd. Site-specific mutation of acidic Fd residues in these regions and the resulting analyses of electron transfer activity and affinity chromatography of those mutants revealed that two acidic regions (a region including Asp26, Glu29 and Glu34, and the other including Asp65 and Glu66) dominantly contribute to the electrostatic interaction with P. falciparum FNR. The combination of Asp26/Glu29/Glu34 conferred a larger contribution than that of Asp65/Glu66, and among Asp26, Glu29 and Glu34, Glu29 was. shown to be the most important residue for the interaction with P. falciparum FNR. These findings provide the basis for understanding molecular recognition between Fd and FNR of the malaria parasite.

DOI： 10.1093/jb/mvm184

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The blockage of transcription elongationby RNApolymerase II (RNAPII) atDNAlesions on the transcribed strand is a serious challenge to accurate transcription. Transcription- coupled DNA repair (TCR), which is assumed to be initiated by the blockage of transcription, rapidly removes lesions on the transcribed strand of expressed genes and allows the resumption of transcription. Although helix- distorting bulky damage such as a cyclobutane pyrimidine dimer is known to block transcription elongation and to be repaired by TCR, it is not clear whether oxidative DNA lesions are repaired by TCR. First, we examined whether transcription elongation by RNAPII is stalled at sites of 2-hydroxyadenine (2-OH-A), 8-oxoadenine (8-oxoA), 8-oxoguanine (8-oxoG), or thymine glycol (Tg) on the transcribed strand. Our results indicate that RNAPII incorporated nucleotides opposite the lesions and then stalled. In addition, we found that transcription elongation factor TFIIS (SII) enabled RNAPII to bypass 8-oxoG but not the other types of damage, while transcription initiation and elongation factor TFIIF did not bypass 8-oxoG. These results suggest that SII is important for preventing cellular death due to oxidative DNA damage, assisting RNAPII to bypass 8-oxoG. (c) 2007 Elsevier B.V All rights reserved.

DOI： 10.1016/j.dnarep.2007.01.014

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD, values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD, values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD, 8.0, the daytime pH in chloroplasts, than at pD, 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

DOI： 10.1074/jbc.m608417200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

DOI： 10.1007/s10858-006-9058-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

For high-throughput protein structural analyses, it is essential to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones involving Escherichia coli cells, have been developed, the number of overexpressed proteins exhibiting the same biological activities as those of the native ones is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the synthesized proteins that should function in solution were found to be in soluble forms. This suggests the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we developed a selective labeling technique for amino acids having amide functional groups (other than proline residues) involving the use of several inhibitors for transaminases. This paper in turn describes a proline-selective labeling technique. Based on our results, we have succeeded in constructing a complete amino acid selective labeling technique for the wheat germ cell-free protein synthesis system.

DOI： 10.1093/jb/mvj174

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The recent explosion in genome sequencing has revealed the great diversity of the cadherin superfamily. Within the superfamily, protocadherins, which are expressed mainly in the nervous system, constitute the largest subgroup. Nevertheless, the structures of only the classical cadherins are known. Thus, to broaden our understanding of the adhesion repertoire of the cadherin superfamily, we determined the structure of the N-terminal first extracellular cadherin domain of the cadherin-related neuronal receptor/protocadherin-alpha 4. The hydrophobic pocket essential for homophilic adhesiveness in the classical cadherins was not found, and the functional significance of this structural domain was supported by exchanging the first extracellular cadherin domains of protocadherin and classical cadherin. Moreover, potentially crucial variations were observed mainly in the loop regions. These included the protocadherin-specific disulfide-bonded Cys-X-5-Cys motif, which showed Ca2+-induced chemical shifts, and the RGD motif, which has been suggested to be involved in heterophilic cell adhesion via the active form of beta 1 integrin. Our findings reveal that the adhesion repertoire of the cadherin superfamily is far more divergent than would be predicted by studying the classical cadherins alone.

DOI： 10.1074/jbc.m603298200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

DOI： 10.1007/s10858-006-9043-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JOHN WILEY & SONS LTD  

Orexins-A and 13, also called hypocretins-1 and 2, respectively, are neuropeptides that regulate feeding and sleep-wakefulness by binding to two orphan G protein-coupled receptors named orexin-1 (OX1R) and orexin-2 (OX2R). The sequences and functions of orexins-A and B are similar to each other, but the high sequence homology (68%) is limited in their C-terminal half regions (residues 15-33). The sequence of the N-terminal half region of orexin-A (residues 1-14), containing two disulfide bonds, is very different from that of orexin-B. The structure of orexin-A was determined using two-dimensional homonuclear and N-15 and C-13 natural abundance heteronuclear NMR experiments. Orexin-A had a compact conformation in the N-terminal half region, which contained a short helix (III: Cys6-Gln9) and was fixed by the two disulfide bonds, and a helix-turn-helix conformation (I: Leu16-Ala23 and II: Asn25-Thr32) in the remaining C-terminal half region. The C-terminal half region had both hydrophobic and hydrophilic residues, which existed on separate surfaces to provide an amphipathic character in helices I and II. The nine residues on the hydrophobic surface are also well conserved in orexin-B, and it was reported that the substitution of each of them with alanine resulted in a significant drop in the functional potency at the receptors. Therefore, we suggest that they form the surface responsible for the main hydrophobic interaction with the receptors. On the other hand, the residues on the hydrophilic surface, together with the hydrophilic residues in the N-terminal half region that form a cluster, are known to make only small contributions to the binding to the receptors through similar alanine-scan experiments. However, since our structure of orexin-A showed that large conformational and electrostatical differences between orexins-A and B were rather concentrated in the N-terminal half regions, we suggest that the region of orexin-A is important for the preference for orexin-A of OX1R. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

DOI： 10.1002/psc.747

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

Invasive potentials of carcinomas greatly contribute to their metastasis, which is a major threat in most cancers. We have recently shown that Arf6 plays a pivotal role in breast cancer invasive activities and identified AMAP1 as an effector of GTP-Arf6 in invasion. Expression of AMAP1 correlates well with invasive phenotypes of primary tumors of the human breast. We also have shown that AMAP1 functions by forming a trimeric protein complex with cortactin and paxillin. In this complex, AMAP1 binds to the src homology 3 (SH3) domain of cortactin via its proline-rich peptide, SKKRPPPPPPGHKRT. SH3 domains are known to bind generally to the proline-rich ligands with a one-to-one stoichiometry. We found that AMAP1/cortactin binding is very atypical in its stoichiometry and interface structure, in which one AMAP1 proline-rich peptide binds to two cortactin SH3 domains simultaneously. We made a cell-permeable peptide derived from the AMAP1 peptide, and we show that this peptide specifically blocks AMAP1/cortactin binding, but not other canonical SH3/proline bindings, and effectively inhibits breast cancer invasion and metastasis. Moreover, this peptide was found to block invasion of other types of cancers, such as glioblastomas and lung carcinomas. We also found that a small-molecule compound, UCS15A, which was previously judged as a weak inhibitor against canonical SH3/proline bindings, effectively inhibits AMAP1/cortactin binding and breast cancer invasion and metastasis. Together with fine structural analysis, we propose that the AMAP1/cortactin complex, which is not detected in normal mammary epithelial cells, is an excellent drug target for cancer therapeutics.

DOI： 10.1073/pnas.0509166103

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

The effects of thermal treatments on the rehydration process and photocatalytic activity were investigated by H-1 NMR spectroscopy for six anatase abundant TiO2 photocatalysts with different properties. Acetic acid and benzoic acid were employed for photodecomposition in aqueous suspension. After the calcinations at 973 K, physisorbed water layers recovered relatively fast for P25, F4, and AMT-600 (shorter than 24 h) with no significant enhancement of the photocatalytic decomposition. On the other hand, for ST-01, UV-100, and AMT-100, the recovery was very slow (longer than 1 week) and only partially reversible, and the photocatalytic decomposition was considerably enhanced but retarded with rehydration. In the presence of adsorbed water, the binding of a carboxyl group of the molecules with adsorbed water is considered to compete with the direct adsorption on the surface, which reduces the amount of the direct adsorption and results in the reduction in the photocatalytic efficiency. In addition, the photocatalytic decomposition of benzoic acid with an aromatic ring was much faster in all of the TiO2 aqueous suspensions and more enhanced for the fully dehydroxylated TiO2 than that of acetic acid. These results suggest that the most efficient photocatalytic sites should be the hydrophobic sites on the TiO2 surface. The difference among the rehydration rates of different TiO2 is discussed in terms of thermally induced changes of surface morphology.

DOI： 10.1021/jp060894v

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

Chitinase C from Streptomyces griseus HUT6037 was discovered as the first bacterial chitinase in family 19 other than chitinases found in higher plants. Chitinase C comprises two domains: a chitin-binding domain (ChBD(ChiC)) for attachment to chitin and a chitin-catalytic domain for digesting chitin. The structure of ChBD(ChiC) was determined by means of C-13-, N-15-, and H-1-resonance nuclear magnetic resonance (NMR) spectroscopy. The conformation of its backbone comprised two P-sheets composed of two and three antiparallel beta-strands, respectively, this being very similar to the backbone conformations of the cellulose-binding domain of endoglucanase Z from Erwinia chrysanthemi (CBDEGZ) and the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12 (ChBD(ChiA1)). The interaction between ChBD(ChiC) and hexa-N-acetyl-chitohexaose was monitored through chemical shift perturbations, which showed that ChBDChiC interacted with the substrate through two aromatic rings exposed to the solvent as CBDEGZ interacts with cellulose through three characteristic aromatic rings. Comparison of the conformations of ChBD(ChiA1), ChBD(ChiC), and other typical chitin- and cellulose-binding domains, which have three solvent-exposed aromatic residues responsible for binding to polysaccharides, has suggested that they have adopted versatile binding site conformations depending on the substrates, with almost the same backbone conformations being retained.

DOI： 10.1093/jb/mvj062

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme cytochiome c(3) (cyt c(3)) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The K-d for the single-site binding was 8.0 mu M, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly N-15-labeled and specifically [N-15]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt c(3) could conduct redox reactions. In light of triheme cytochrome c(7), the CO-bound cyt c(3) may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H-2 cycling. Therefore, the CO-bound cyt c(3) may play a role in maintaining electron transport pathways on accumulation of toxic CO for its utilization.

DOI： 10.1021/bi051867i

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.

DOI： 10.1074/jbc.m510530200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

in FoF1-ATP synthase, an oligomer ring of F(o)c subunits acts as a rotary proton channel of the F-o-proton motor. On the basis of the solution structure of the Escherichia coli F(o)c (EF(o)c) monomer, the rotation of the C-terminal helix coupled with the reorientation of the essential Asp61 side-chain on deprotonation was proposed to drive rotation of the whole c-ring. We have determined the NMR structure of F(o)c from thermophilic Bacillus PS3, TF(o)c, in an organic solvent mixture (chloroform/methanol (3:1, v/v)). Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TFoc protrudes toward the outside of the hairpin, a third orientation that differs from either of the two orientations in EF(o)c. Therefore, it would be inappropriate to draw conclusions about the mechanism of c-ring rotation on the basis of the conformations observed only for EF(o)c. The appearance of different hairpin structures shows that there are multiple energy minima for the hairpin structure in terms of helix rotation and axial displacement. The multiple energy minima may also provide a base for the different oligomeric states in the c-ring structure. A rotation mechanism of the F-o motor coupled with H+-translocation is discussed on the basis of these results and the recently reported crystal structure of the c-ring from Ilyobacter tartaricus Na+-ATPase. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2006.01.011

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Ferredoxin-NADP(+) reductase (FNR) catalyzes the reduction of NADP(+) through the formation of an electron transfer complex with ferredoxin. To gain insight into the interaction of this enzyme with substrates at both ends of the polypeptide chain, we performed NMR analyses of a 314-residue maize leaf FNR with a nearly complete assignment of the backbone resonances. The chemical shift perturbation upon formation of the complex indicated that a flexible N-terminal region of FNR contributed to the interaction with maize ferredoxin, and an analysis of N-terminally truncated mutants of FNR confirmed the importance of this region for the binding of ferredoxin. Comparison between the spectra of FNR in the NADP(+)- and inhibitor-bound states also revealed that the nicotinamide moiety of NADP(+) was accessible to the C-terminal Tyr314. We propose that the formation of the catalytic competent complex of FNR and substrates is achieved through the interaction of the N- and C-terminal segments with ferredoxin and NADP(+), respectively. Since the ends of the polypeptide chain act as flexible regions of proteins, they may contribute to the search of a larger space for a binding partner and to the opening of active sites.

DOI： 10.1021/bi050424b

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

DOI： 10.1007/s10858-005-2450-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING LTD  

The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far-UV and near-UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes alpha-helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side-chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB N-15-H-1 heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.

DOI： 10.1111/j.1742-4658.2004.04513.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Although more than 100 mutations have been identified in the copper/zinc superoxide dismutase (Cu/Zn-SOD) in familial amyotrophic lateral sclerosis (FAILS), the mechanism responsible for FAILS remains unclear. The finding of the present study shows that FALS-causiing mutant Cu/Zn-SOD proteins (FALS mutant SODs), but not wild-type SOD, are barely detected by three monoclonal antibodies (mAbs) in Western blot analyses. The enzyme-linked immunosorbent assay for denatured FALS mutant SODs by dithiothreitol, SDS, or heat treatment also showed a lowered immunoreactivity against the mAbs compared with wild-type SOD. Because all the epitopes of these mAbs are mapped within the Greek key loop (residues 102-115 in human Cu/Zn-SOD), these data suggest that different conformational changes occur in the loop between wild-type and FAILS mutant SODs during the unfolding process. Circular dichroism measurements revealed that the FALS mutant SODs are sensitive to denaturation by dithiothreitol, SDS, or heat treatment, but these results do not completely explain the different recognition by the mAbs between wild-type and FAILS mutant SODs under the denatured conditions. The study on the conformational changes in local areas monitoring with mAbs may provide a new insight into the etiology of FALS.

DOI： 10.1074/jbc.m406106200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5- bisphosphate (PI(4,5) P-2), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5) P-2 in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5) P-2-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein - protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

DOI： 10.1038/sj.emboj.7600442

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：KLUWER ACADEMIC PUBL  

For understanding the precise mechanisms of molecular recognition of proteins, three-dimensional structural analyses of the protein-protein complexes are essential. For this purpose, a new method to reveal complex structures was developed with the assistance of saturation transfer (SAT) and residual dipolar coupling (RDC) by heteronuclear NMR experiments, without any paired intermolecular NOE information. The SAT and RDC experiments provide the information of the interfacial residues and the relative orientations of the two protein molecules, respectively. Docking simulation was then made to reconstruct a complex conformation, which satisfies the SAT and RDC data. The method was applied to the CAD-ICAD complex structure, which was previously determined by the NOE-distance geometry method. The quality of the current model was evaluated.

DOI： 10.1023/b:jnmr.0000032613.05864.87

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：KLUWER ACADEMIC PUBL  

A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual N-15, H-1-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.

DOI： 10.1023/b:jnmr.0000032611.72827.de

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The cis-syn cyclobutane pyrimidine dimer (CPD) is a cytotoxic, mutagenic and carcinogenic DNA photoproduct and is repaired by the nucleotide excision repair (NER) pathway in mammalian cells. The XPC-hHR23B complex as the initiator of global genomic NER binds to sites of certain kinds of DNA damage. Although CPDs are rarely recognized by the XPC-hHR23B complex, the presence of mismatched bases opposite a CPD significantly increased the binding affinity of the XPC-hHR23B complex to the CPD. In order to decipher the properties of the DNA structures that determine the binding affinity for XPC-hHR23B to DNA, we carried out structural analyses of the various types of CPDs by NMR spectroscopy. The DNA duplex which contains a single 3' T.G wobble pair in a CPD (CPD/GA duplex) induces little conformational distortion. However, severe distortion of the helical conformation occurs when a CPD contains double T.G wobble pairs (CPD/GG duplex) even though the T residues of the CPD form stable hydrogen bonds with the opposite G residues. The helical bending angle of the CPD/GG duplex was larger than those of the CPD/GA duplex and properly matched CPD/AA duplex. The fluctuation of the backbone conformation and significant changes in the widths of the major and minor grooves at the double T.G wobble paired site were also observed in the CPD/GG duplex. These structural features were also found in a duplex that contains the (6-4) adduct, which is efficiently recognized by the XPC-hHR23B complex. Thus, we suggest that the unique structural features of the DNA double helix (that is, helical bending, flexible backbone conformation, and significant changes of the major and/or minor grooves) might be important factors in determining the binding affinity of the XPC-hHR23B complex to DNA.

DOI： 10.1093/nar/gkh568

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

A novel type of calixsugars, containing sugar moieties at the methine bridges of the calixarene is introduced. These calixsugars were prepared via a nonconvergent stepwise fragment condensation. Four new stereogenic centers were formed simultaneously, and only one diastereoisomer 5 was isolated. The condensation procedure is remarkably mild allowing for a large diversity of labile groups to be used. The solution structure of calixarene 5 with two D-glucose and two hexyl moieties was determined by NMR spectroscopy by means of NOEs and coupling constants for molecular dynamics (MD). Chemical shifts were used to validate the conformation with the least NOE and J violations. The structure of calixsugar 5 has the configuration rctc referring to one sugar residue (r = reference, c = cis, t = trans) and adopts a diamond conformation for the macrocyclic backbone with the two sugar moieties axial on opposite sides of the macrocyclic ring and the two hexyl groups on the same side.

DOI： 10.1002/1522-2675(200211)85:11<3895::AID-HLCA3895>3.0.CO;2-2

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Growing evidence suggests that horizontal gene transfer plays an integral role in the evolution of bacterial genomes. One of the debated examples of horizontal gene transfer from animal to prokaryote is the fibronectin type III domain (FnIIID). Certain extracellular proteins of soil bacteria contain an unusual cluster of FnHIDs, which show sequence similarity to those of animals and are likely to have been acquired horizontally from animals. Here we report the solution structure of the FnIIID of chitinase A1 from Bacillus circulans WL-12. To the best of our knowledge, this is the first tertiary structure to be reported for an FnIIID from a bacterial protein. The structure of the domain shows significant similarity to FnIIIDs from animal proteins. Sequence comparisons with FnIIIDs from other soil bacteria proteins show that the core-forming residues are highly conserved and, thus, are under strong evolutionary pressure. Striking similarities in the tertiary structures of bacterial FnIlIDs and their mammalian counterparts may support the hypothesis that the evolution of the FnIIID in bacterial carbohydrases occurred horizontally. The total lack of surface-exposed aromatic residues also suggests that the role of this FnIIID is different from those of other bacterial beta-sandwich domains, which function as carbohydrate-binding modules.

DOI： 10.1074/jbc.m109726200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

In vertebrates, the biological consequences of DNA methylation are often mediated by protein factors containing conserved methyl-CpG binding domains (MBDs). Mutations in the MBD protein MeCP2 cause the neurodevelopmental disease Rett syndrome. We report here the solution structure of the MBD of the human methylation-dependent transcriptional regulator MBD1 bound to methylated BRA. DNA binding causes a loop in MBD1 to fold into a major and novel DNA binding interface. Recognition of the methyl groups and CG sequence at the methylation site is due to five highly conserved residues that form a hydrophobic patch. The structure indicates how MBD may access nucleosomal DNA without encountering steric interference from core histones, and provides a basis to interpret mutations linked to Rett syndrome in MeCP2.

DOI： 10.1016/s0092-8674(01)00324-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE AMERICA INC  

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(I-ZI)NH2, bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3-7 form a unique beta -coil structure that is preceded by an N-terminal extended tail. This beta -coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8-21) forms an ct-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

DOI： 10.1038/84159

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

An Escherichia coli sensor kinase, ArcB, transfers a phosphoryl group to a partner response regulator in response to anaerobic conditions. Multidimensional NMR techniques were applied to determine the solution structure of the histidine-containing phosphotransfer signaling domain of ArcB (HPtArcB), which has a phosphorylation site, His717. The backbone dynamics were also investigated by analyses of the N-15 relaxation data and amide hydrogen exchange rates. Furthermore, the protonation states of the histidine imidazole rings were characterized by means of H-1 and N-15 chemical shifts at various pHs. The determined solution structure of HPtArcB contains five helices and forms a four-helix bundle motif like other HPt domains. The obtained order parameters, S-2, {H-1}-N-15 heteronuclear NOE values, and chemical exchange parameters, R-ex, showed that the a-helical regions of HPtArcB are rigid on both picosecond to nanosecond and microsecond to millisecond time scales. On the other hand, helix D, which contains His717, exhibited low protection factors of less than 4000, indicating the presence of fluctuations on a slower time scale in helix D. These results suggest that HPtArcB may undergo a small conformational change in helix D upon phosphorylation. It was also shown that the imidazole ring of His717 has a pK(a) value of 6.76, which is similar to that of a solvent-exposed histidine imidazole ring, and that a pair of deprotonated neutral tautomers are rapidly exchanged with each other. This is consistent with the solution structure of HPtArcB, in which the imidazole ring of His717 is exposed to the solvent.

DOI： 10.1021/bi001619g

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Chitinase Al from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD(Chia1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(Chia1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(Chia1) was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD(Chia1) to regenerated chitin. These observations suggest that ChBD(Chia1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(Chia1) exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pi and by the presence of NaCl, suggesting that the binding of ChBD(Chia1) is mediated mainly by hydrophobic interactions. Hydrolysis of beta-chitin microcrystals by intact chitinase Al and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta-chitin microcrystals but greatly enhances the efficiency of degradation.

DOI： 10.1128/jb.182.11.3045-3054.2000

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/ja000005w

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The three-dimensional structure of the chitin-binding domain (ChBD) of chitinase A1 (ChiA1) from a Grampositive bacterium, Bacillus circulans WL-12, was determined by means of multidimensional heteronuclear NMR methods. ChiA1 is a glycosidase that hydrolyzes chitin and is composed of an N-terminal catalytic domain, two fibronectin type Iii-like domains, and C-terminal ChBD(ChiA1), (45 residues, Ala(655)-Gln(699)), which binds specifically to insoluble chitin, ChBD(ChiA1) has a compact and globular structure with the topology of a twisted beta-sandwich, This domain contains two antiparallel beta-sheets, one composed of three strands and the other of two strands. The core region formed by the hydrophobic and aromatic residues makes the overall structure rigid and compact. The overall topology of ChBD(ChiA1) is similar to that of the cellulose-binding domain (CBD) of Erwinia chrysanthemi endoglucanase Z (CBDEGZ). However, ChBD(ChiA1) lacks the three aromatic residues aligned linearly and exposed to the solvent, which probably interact with cellulose in CBDs. Therefore, the binding mechanism of a group of ChBDs including ChBD(ChiA1) may be different from that proposed for CBDs.

DOI： 10.1074/jbc.275.18.13654

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：KLUWER ACADEMIC PUBL  

Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator that is thought to bridge between the TATA box-binding protein (TBP) and DNA binding regulatory factors, and is conserved from yeast to human. Human MBF1 (hMBF1) can bind to TBP and to the nuclear receptor Ad4BP, and is suggested to mediate Ad4BP-dependent transcriptional activation. Here we report the resonance assignments and secondary structure of hMBF1 (57-148) that contains both TBP binding and activator binding residues. N-15 relaxation data were also obtained. As a result, hMBF1 (57-148) was shown to consist of flexible N-terminal residues and a C-terminal domain. The C-terminal domain contains four helices and a conserved C-terminal region.

DOI： 10.1023/a:1008347729176

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL SCIENCE LTD  

Background: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP.
Results: The two-dimensional (2D) H-1-N-15 correlation spectrum of N-15 labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by alpha-chymotrypsin yielded a similar to 9kDa fragment. N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1(CTD). As previous deletion analyses have shown that MBF1(CTD), is capable of binding to TBP, it is suggested that MBF1(CTD) is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of three-dimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1(CTD), was determined. As a result, MBF1(CTD) was shown to contain four amphipathic helices and a conserved C-terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix.
Conclusions: Structural analyses revealed that MBF1 consists of two structurally different domains. A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1(CTD). MBF1(CTD) is made up of four amphipathic helices and conserved C-terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.

DOI： 10.1046/j.1365-2443.1999.00267.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

XPA is involved in the damage recognition step of nucleotide excision repair (NER), XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA), The domain consists of a zinc-containing subdomain with a compact globular structure and a C-terminal subdomain with a positively charged cleft in a novel alpha/beta structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods, N-15 relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion, In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (D-parallel to/D-perpendicular to) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-terminal subdomain, This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA.

DOI： 10.1093/oxfordjournals.jbchem.a022313

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE AMERICA INC  

The solution structure of the central domain of the human nucleotide excision repair protein XPA, which binds to damaged DNA and replication protein A (RPA), Tvas determined by nuclear magnetic resonance (NMR) spectroscopy. The central domain consists of a zinc-containing subdomain and a C-terminal subdomain. The zinc-containing subdomain has a compact globular structure and is distinct from the zinc-fingers found in transcription factors. The C-terminal subdomain folds into a novel alpha/beta structure with a positively charged superficial deft. From the NMR spectra of the complexes, DNA and RPA binding surfaces are suggested.

DOI： 10.1038/1400

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1006/jmre.1996.7497

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記述言語：日本語   出版者・発行元：日本光合成研究会  

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.59.032

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.59.032

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記述言語：日本語   出版者・発行元：日本核磁気共鳴学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.50.S30_2

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記述言語：英語   掲載種別：記事・総説・解説・論説等（国際会議プロシーディングズ）   出版者・発行元：Springer  

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記述言語：日本語   出版者・発行元：大阪大学低温センター  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL PUBLISHING  

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記述言語：日本語   出版者・発行元：日本植物生理学会  

電子伝達タンパク質であるフェレドキシン(Fd)は葉緑体中に存在し様々なFd依存性酵素に電子伝達複合体を形成することで電子を供与している。本研究ではFdとFd依存性酵素の一つである亜硫酸還元酵素(SiR)との相互作用様式及び電子伝達メカニズムを解明することを目的とした。まず、FdのSiRとの相互作用領域を明らかにするために<SUP>15</SUP>NラベルされたFdを用いNMRスペクトルによる化学シフト摂動法の実験を行った。その結果、大きな化学シフトの変化を起こす、すなわち相互作用に関与していると考えられる領域が主に負電荷を持つ二箇所の領域において見られた。この領域の相互作用における重要性はFdの部位特異的変異体を用い確認した。次に、SiR側のFdとの相互作用領域と電子伝達メカニズムを解明するために、相互作用及び電子伝達に関与していると予測されるアミノ酸残基を改変した数種のSiRの部位特異的変異体を作製し、酵素活性及びNMR測定を行った。その結果、それぞれの実験において野生型と比較し興味深い変化を示す変異体が存在した。今回の実験で得られた最も重要な所見の一つはFdのSiRに対する相互作用領域がこれまでに報告されているNADP<SUP>+</SUP>還元酵素との相互作用領域と異なっていた点である。すなわちFdはそれぞれのFd依存性酵素によって相互作用領域を使い分けていることを示している。

DOI： 10.14841/jspp.2005.0.253.0

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.45.S208_4

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：日本語   出版者・発行元：社団法人 日本分光学会  

DOI： 10.5111/bunkou.53.259