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

Protein aggregation is a major biomedical challenge necessitating effective protein stabilization strategies. However, the specific interactions between hydrophobic components and proteins in relation to protein aggregation remain poorly understood. Here, we investigate the molecular mechanism of protein aggregation inhibition using zwitterionic polymers, revealing their remarkable efficiency in suppressing protein aggregation. Higher molecular weight and increased hydrophobic modification greatly contribute to near-complete inhibition. Comprehensive investigations employing various techniques unravel weak and reversible interactions between sulfobetaine polymers and proteins, amplified by increasing hydrophobicity and molecular weight. The polymers serve as reversible molecular shields, disrupting the aggregation pathway and impeding the formation of highly aggregation-prone intermediates. Upon stress removal, partially unfolded intermediates regain their native states through refolding. This study represents a comprehensive exploration of protein stabilization with zwitterionic polymers, yielding valuable insights into protein denaturation, the aggregation pathway, and protein-folding dynamics. These findings hold potential for advancing protein therapeutics.

DOI： 10.1016/j.xcrp.2024.102012

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

Aquaporin (Aqp) 10 is a member of the aquaglyceroporin subfamily of water channels, and human Aqp10 is permeable to solutes such as glycerol, urea, and boric acid. Tetrapods have a single aqp10 gene, whereas ray-finned fishes have paralogs of this gene through tandem duplication, whole-genome duplication, and subsequent deletion. A previous study on Aqps in the Japanese pufferfish Takifugu rubripes showed that one pufferfish paralog, Aqp10.2b, was permeable to water and glycerol, but not to urea and boric acid. To understand the functional differences of Aqp10 s between humans and pufferfish from an evolutionary perspective, we analyzed Aqp10 s from an amphibian (Xenopus laevis) and a lobe-finned fish (Protopterus annectens), and Aqp10.1 and Aqp10.2 from several ray-finned fishes (Polypterus senegalus, Lepisosteus oculatus, Danio rerio and Clupea pallasii). The expression of tetrapod and lobe-finned fish Aqp10 s and Aqp10.1-derived Aqps in ray-finned fishes in Xenopus oocytes increased the membrane permeabilities to water, glycerol, urea and boric acid. In contrast, Aqp10.2-derived Aqps in ray-finned fishes increased water and glycerol permeabilities, while those of urea and boric acid were much weaker than those of Aqp10.1-derived Aqps. These results indicate that water, glycerol, urea, and boric acid permeability are plesiomorphic activities of Aqp10 s, and that the ray-finned fish-specific Aqp10.2 paralogs have secondarily reduced or lost urea and boric acid permeability.

DOI： 10.1093/gbe/evad221

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Luciferases are widely used as reporter proteins in various fields. Recently, we developed a minimal bright luciferase, picALuc, via partial deletion of the artificial luciferase (ALuc) derived from copepods luciferases. However, the structures of copepod luciferases in the substrate-bound state remain unknown. Moreover, as suggested by structural modeling, picALuc has a larger active site cavity, unlike that in other copepod luciferases. Here, to explore the bioluminescence mechanism of picALuc and its luminescence properties, we conducted multiple mutational analyses, and identified residues and regions important for catalysis and bioluminescence. Mutations of residues likely involved in catalysis (S33, H34, and D55) markedly reduced bioluminescence, whereas that of residue (E50) (near the substrate in the structural model) enhanced luminescence intensity. Furthermore, deletion mutants (Δ70–Δ78) in the loop region (around I73) exhibited longer luminescence lifetimes (~30 min), and were reactivated multiple times upon re-addition of the substrate. Due to the high thermostability of picALuc, one of its representative mutant (Δ74), was able to be reused, i.e., luminescence recycling, for day-scale time at room temperature. These findings provide important insights regarding picALuc bioluminescence mechanism and copepod luciferases, and may help with sustained observations in a variety of applications.

DOI： 10.1111/febs.16937

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Luciferases are widely used as reporter proteins in diverse fields from basic biology to medical and environmental researches. Development of luciferase applications for reporter proteins requires small size without target inhibition, appropriate genomic insertion for high expression level, and bright emission for detection sensitivity. We previously developed the minimal luciferase picALuc, but its luminescence was still dim compared to other bright luciferases in terms of expression in E. coli. In this study, diverse additions of oligopeptides with charged residues (eight amino acids in total) to the C-terminus of picALuc enhanced luminescence by up to approximately 50-fold, i.e., enhanced enzymatic activity. Moreover, these high luminescence activities were achieved in bacterial and mammalian expression, suggesting their further applicability in many expression systems. The finding in this study that the simple addition of oligopeptides with charged residues (or charge engineering of this kind) enhances enzymatic activity may be applied to a wide variety of enzymatic reactions and protein functions.

DOI： 10.1002/biot.202200560

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

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E. coli. Furthermore, we determined that transcription of trkG in E. coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E. coli during evolution as a K+ uptake system that coexists with TrkH.

DOI： 10.1016/j.jbc.2022.102846

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Classical cadherins play key roles in cell-cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

DOI： 10.1073/pnas.2208067119

PubMed

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

Luciferases are widely used as sensitive reporters in various fields ranging from basic biology to medical diagnosis, public health, and food inspection. Scientists have isolated novel luciferases from bioluminescent organisms and concentrated on improving their brightness and thermostability. Recently, small bright luciferases such as artificial luciferase (ALuc) (21 kDa), NanoLuc (19 kDa), GLuc (18 kDa), and TurboLuc (16 kDa) have been reported. However, smaller, brighter, and more stable luciferases are desired for further applications. Here, we constructed the smallest and bright mutant of ALuc, named “picALuc” (13 kDa). picALuc retained the luminescence activity of the full-length ALuc; moreover, its brightness and thermostability were at the same levels as NanoLuc. Furthermore, we showed the advantage of picALuc for the bioluminescence resonance energy transfer-based assay due to its smallness. Our development has opened the door for wider and more practical applications of luciferases.

DOI： 10.1021/acschembio.1c00897

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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-VCH  

The dynamic process of formation of protein assemblies is essential to form highly ordered structures in biological systems. Advances in structural and synthetic biology have led to the construction of artificial protein assemblies. However, development of design strategies exploiting the anisotropic shape of building blocks of protein assemblies has not yet been achieved. Here, the 2D assembly pattern of protein needles (PNs) is controlled by regulating their tip-to-tip interactions. The PN is an anisotropic needle-shaped protein composed of β-helix, foldon, and His-tag. Three different types of tip-modified PNs are designed by deleting the His-tag and foldon to change the protein–protein interactions. Observing their assembly by high-speed atomic force microscopy (HS-AFM) reveals that PN, His-tag deleted PN, and His-tag and foldon deleted PN form triangular lattices, the monomeric state with nematic order, and fiber assemblies, respectively, on a mica surface. Their assembly dynamics are observed by HS-AFM and analyzed by the theoretical models. Monte Carlo (MC) simulations indicate that the mica-PN interactions and the flexible and multipoint His-tag interactions cooperatively guide the formation of the triangular lattice. This work is expected to provide a new strategy for constructing supramolecular protein architectures by controlling directional interactions of anisotropic shaped proteins.

DOI： 10.1002/smll.202106401

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/smll.202106401

担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Portland Press Ltd.  

The biological activities of living organisms involve various inputs and outputs. The ATP-driven substances (biomolecules) responsible for these kinds of activities through membrane (i.e. uptake and efflux of substrates) include ATP-binding cassette (ABC) transporters, some of which play important roles in multidrug resistance. The basic architecture of ABC transporters comprises transmembrane domains (TMDs) and nucleotide-binding domains (NBDs). The functional dynamics (substrate transport) of ABC transporters are realized by concerted motions, such as NBD dimerization, mechanical transmission via coupling helices (CHs), and the translocation of substrates through TMDs, which are induced by the binding and/or hydrolysis of ATP molecules and substrates. In this mini-review, we briefly discuss recent progresses in the structural dynamics as revealed by molecular simulation studies at all-atom (AA), coarse-grained (CG), and quantum mechanics/molecular mechanics (QM/MM) levels.

DOI： 10.1042/bst20200710

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Monoclonal antibodies (mAbs) are attractive therapeutics for treating a wide range of human disorders, and bind to the antigen through their complementarity-determining regions (CDRs). Small stable proteins containing structurally retained CDRs are promising alternatives to mAbs. In this report, we present a method to create such proteins, named fluctuation-regulated affinity proteins (FLAPs). Thirteen graft acceptor (GA) sites that efficiently immobilise the grafted peptide structure were initially selected from six small protein scaffolds by computational identification. Five CDR peptides extracted by binding energy calculations from mAbs against breast cancer marker human epithelial growth factor receptor type 2 (HER2) were then grafted to the selected scaffolds. The combination of five CDR peptides and 13 GA sites in six scaffolds revealed that three of the 65 combinations showed specific binding to HER2 with dissociation constants (KD) of 270-350 nM in biolayer interferometry and 24-65 nM in ELISA. The FLAPs specifically detected HER2-overexpressing cancer cells. Thus, the present strategy is a promising and practical method for developing small antibody mimetics.

DOI： 10.1038/s41598-020-57713-4

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nanosheets have thicknesses on the order of nanometers and planar dimensions in the micrometer range. Nanomaterials that are capable of converting reversibly between 2D nanosheets and 3D structures in response to specific triggers can enable construction of nanodevices. Supra‐molecular lipid nanosheets and their triggered conversions to 3D structures including vesicles and cups are reported. They are produced from lipid vesicles upon addition of amphiphilic peptides and cationic copolymers that act as peptide chaperones. By regulation of the chaperoning activity of the copolymer, 2D to 3D conversions are reversibly triggered, allowing tuning of lipid bilayer structures and functionalities. 3D lipid vesicles are converted to 2D lipid nanosheets by addition of anionic amphiphilic peptides and cationic copolymers that act as peptide chaperones. 2D–3D reconversion is triggered by stimuli such as enzymatic activity or pH change, allowing tuning of lipid bilayer structures and functionalities.

DOI： 10.1002/adma.201904032

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mechanosensitive channels play an important role in the adaptation of cells to hypo-osmotic shock. Among members of this channel family in Escherichia coli, the exact function and physiological role of the mechanosensitive channel homolog YbdG remain unclear. Characterization of YbdG's physiological role has been hampered by its lack of measurable transport activity. Using a nitrosoguanidine mutagenesis-aided screen in combination with next-generation sequencing, here we isolated a mutant with a point mutation in ybdG This mutation (resulting in a I167T change) conferred sensitivity to high osmotic stress, and the mutant cells differed from WT cells in morphology during hyperosmotic stress at alkaline pH. Interestingly, unlike the cells containing the I167T variant, a null-ybdG mutant did not exhibit this sensitivity and phenotype. Although I167T was located near the putative ion-conducting pore in a transmembrane region of YbdG, no change in ion channel activities of YbdG-I167T was detected. Of note, introduction of the WT C-terminal cytosolic region of YbdG into the I167T variant complemented the osmo-sensitive phenotype. Co-precipitation of proteins interacting with the C-terminal YbdG region led to the isolation of HldD and FbaA, whose overexpression in cells containing the YbdG-I167T variant partially rescued the osmo-sensitive phenotype. This study indicates that YbdG functions as a component of a mechanosensing system that transmits signals triggered by external osmotic changes to intracellular factors. The cellular role of YbdG uncovered here goes beyond its predicted function as an ion or solute transport protein.

DOI： 10.1074/jbc.RA118.007340

PubMed

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The biological functions of ATPases, such as myosin, kinesin, and ABC transporter, are due to large conformational motions driven by energy obtained from ATP. Elucidation of the mechanisms underlying these ATP‐driven movements is one of the greatest challenges in computational chemistry. It has been shown that the MARTINI coarse‐grained method is a promising tool for the investigation of large conformational motions in various proteins. However, this method has not yet been applied to ATPases because of the lack of a force field for the ATP molecule. Here, we developed force field parameters for the ATP molecule and conducted simulations using these parameters for the subunits (MalK2) and the full‐length structure (MalFGK2‐E) of a maltose transporter. It was found for both targets that the dimerization of the nucleotide binding domains (NBDs) is induced upon ATP binding. Moreover, for the full‐length transporter, the conformational transition from the pre‐translocation state to the outward‐facing state was observed and was accompanied by an initial transport motion of the substrate. It is expected that coarse‐grained simulations utilizing the parameters for the ATP molecule developed here will serve as a powerful tool for investigating other ATPases as well.

DOI： 10.1002/jcc.25861

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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/jacs.8b10168

Web of Science

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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  

Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s−1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.

DOI： 10.1038/s41467-018-06362-3

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その他リンク： http://www.nature.com/articles/s41467-018-06362-3

担当区分：最終著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-017-16434-x

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.biochem.6b00947

Web of Science

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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/bi500255j

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M113.452623

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/srep00331

Scopus

PubMed

J-GLOBAL

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1529/biophysj.107.115261

Web of Science

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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.jsb.2006.10.006

Scopus

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/sstr.202500501

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Aquaporin (Aqp) 10 is a member of the aquaglyceriporin family, which transports small, uncharged solutes in addition to water. Although the solute selectivity of aquaglyceroporins varies, the mechanism of solute selectivity has not yet been fully elucidated. The common ancestor of ray-finned fish possessed two paralogous genes for aquaporin 10, aqp10.1 and aqp10.2, which produce Aqps with different solute selectivities. Most teleosts possess one or more ohnologs derived from aqp10.1 and aqp10.2; however, the common ancestor of Anguilliformes species lost all aqp10.1-derived ohnologs. Anguilliformes species, except Anguilla species, have one aqp10.2b, but recent tandem duplications in the European eel have generated three aqp10.2b paralogs (aqp10.2b1–aqp10.2b3), whose activities remain ambiguous. In this study, we found that the four sites forming the aromatic/arginine (ar/R) selectivity filter in European eel Aqp10.2b1 were identical to those in Aqp10.2b of other species. However, the Y residue at position 3 was replaced with G in the ar/R selectivity filter of Aqp10.2b2 and b3. When expressed in Xenopus oocytes, Aqp10.2b2 and b3 showed higher permeability to urea and boric acid than Aqp10.2b1, indicating that Aqp10.2b2 and b3 acquired broad solute selectivity similar to that of Aqp10.1, which was lost in the ancestral Anguilliformes species. Urea and boric acid permeabilities of Aqp10.2b1 increased when the Y residue at position 3 of the ar/R selectivity filter was replaced with G. Overall, our results outline the history of the loss and gain of Aqp10 paralogs with broad solute selectivity in anguillid eels.

DOI： 10.1093/gbe/evaf169

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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：American Chemical Society (ACS)  

Nuclear receptors (NRs) play pivotal roles in functionally diverse cell signaling cascades, regulating metabolism and homeostasis. This study introduces a broadly applicable molecular imaging platform for NR activities based on four rationally designed single-chain bioluminescent probes named P1–P4. As all the ligand binding domains (LBDs) of NRs are highly conserved, the probe portfolio was exemplified using the LBD of peroxisome proliferator-activated receptor γ (PPARγ-LBD), i.e., the LBD was centrally positioned and fused with luciferases and/or fluorescent proteins based on the schemes of the protein-fragment complementation assay (PCA), molecular strain (MS) probe, circular permutation (CP) probe, and bioluminescence resonance energy transfer (BRET) system. In the developing process, we identified the optimal dissection sites for the marine luciferase RLuc for PCA systems and validated them in in vitro and in vivo studies in response to various ligands. Among the probes, P3 and P4 series probes exhibited strong BL intensities in response to a PPARγ agonist with signal-to-background ratios of maximally 14-fold. The animal study using furimazine (FMZ) substrate analogs such as Ad-FMZ showed that the probes can sustain agonist-dependent BL signals for up to 24 h in animal models. Considering the biological importance of NRs, the molecular imaging platform with the portfolio of probes developed in this study can contribute to interrogate many NR-related cell signaling pathways by replacing the PPARγ-LBD with LBDs from other NRs in the probes.

DOI： 10.1021/acsomega.5c04665

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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 University Press (OUP)  

Abstract

Escherichia coli TrkG and TrkH transporters contain a unique N-terminal Domain-0 (D0). Our findings reveal that D0 supports both the function and stability of TrkG, enabling K+ and Na+ uptake, whereas it is not essential for TrkH-mediated K+ uptake. This difference can be attributed to D0 role in stabilizing polar residues within TrkG core transmembrane domains.

DOI： 10.1093/bbb/zbaf101

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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  

By heating 1-(ω-azidoalkyl)-2-(2,2-dihalovinyl)arenes inDMF, intramolecular Huisgen cycloaddition betωeen the azido groupand the 1,1-dihalovinyl moiety proceeded smoothly to afford 5-halo-1,2,3-triazole-fused tricyclic benzocondensed compounds.Leveraging the remaining halogen groups, carbon-chain extension viaMizoroki–Heck or Suzuki–Miyaura coupling reactions, folloωed by anintramolecular Friedel–Crafts reaction, furnished polycyclic systemsincorporating fused triazole rings. Furthermore, transformation of thehalogen groups into 2-nitrobenzene derivatives via Suzuki–Miyauracross-coupling, folloωed by a Cadogan reaction usingtriphenylphosphine, provided a series of fluorescent pentacycliccompounds.

DOI： 10.1002/ejoc.202500355

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記述言語：英語  

Molecular imaging probes play a pivotal role in assaying molecular events in various physiological systems. In this study, we demonstrate a new genre of bioluminescent probes for imaging protein–protein interactions (PPIs) in mammalian cells, named the molecular association assay (MAA) probe. The MAA probe is designed to be as simple as a full-length marine luciferase fused to a protein of interest with a flexible linker. This simple fusion protein alone surprisingly works by recognizing a specific ligand, interacting with a counterpart protein of the PPI, and developing bioluminescence (BL) in mammalian cells. We made use of an artificial intelligence (AI) tool to simulate the binding modes and working mechanisms. Our AlphaFold-based analysis on the binding mode suggests that the hinge region of the MAA probe is flexible before ligand binding but becomes stiff after ligand binding and protein association. The sensorial properties of representative MAA probes, FRB-ALuc23 and FRB-R86SG, are characterized with respect to the quantitative feature, BL spectrum, and in vivo tumor imaging using xenografted mice. Our AI-based simulation of the working mechanisms reveals that the association of MAA probes with the other proteins works in a way to facilitate the substrate’s access to the active sites of the luciferase (ALuc23 or R86SG). We prove that the concept of MAA is generally applicable to other examples, such as the ALuc16- or R86SG-fused estrogen receptor ligand-binding domain (ER LBD). Considering the versatility of this conceptionally unique and distinctive molecular imaging probe compared to conventional ones, we are expecting the widespread application of these probes as a new imaging repertoire to determine PPIs in living organisms.

DOI： 10.3390/bios15050299

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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：American Chemical Society (ACS)  

Bioluminescence (BL) is an emerging optical readout that has been extensively used in various bioassays and molecular imaging systems. In this study, we present the bioanalytical application of marine luciferins as an excellent optical indicator for noninvasive imaging of serum albumins. We synthesized 30 kinds of regioisomeric coelenterazine (CTZ) analogs and investigated their specificities for major serum proteins from various species. The results found that some of the CTZ analogs exhibited surprisingly specific optical signals upon binding with the serum albumins. These CTZ analogs showed diverse emission spectra ranging from 495 to 558 nm according to the albumin species used acting as pseudoluciferases. The selective albumin indicators, TS1 and TS2, exhibited long and linear dose-response curves and were sensitive enough to determine clinically normal and abnormal (microalbuminuria) ranges of albumins in saliva and urine. The sensitivity of this assay is superior to that of the conventional Bromocresol purple (BCP) method. We further demonstrated the advantages of the albumin indicators through noninvasive imaging of liver-albumin in vivo in living mice. The in vivo and ex vivo imaging results confirmed that the CTZ analog TS2 can sensitively image the liver-albumin in vivo with high signal-to-background ratio. This study paves a new way to make use of CTZ analogs for noninvasive albumin imaging and conceptualizes the pseudoluciferase-based imaging. The distinct in vivo imaging of serum albumins can potentially aid clinicians in providing insight into patients' liver function and other vital factors needed for whole-body homeostasis.

DOI： 10.1021/acschembio.4c00740

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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  

Coelenterazine (CTZ) is a common substrate of marine luciferases upon emission of bioluminescence (BL) in living organisms. Because CTZ works as a “luminophore” in the process of BL emission, the chemical modification has been centered for improving the optical properties of BL. In this review, we showcase recent advances in CTZ designs with unique functionalities. We first elucidate the light-emitting mechanisms of CTZ, and then focus on how the rational modification of CTZ analogs developed in recent years are connected to the development of unique functionalities even without luciferases, which include color tunability covering the visible region, specificity to various proteins (e.g., luciferase, albumin, and virus protein), and activatability to ions or reactive oxygen species (ROS) and anticancer drugs. This review provides new insights into the broad utilities of CTZ analogs with designed functionalities in bioassays and molecular imaging.

DOI： 10.3390/s25061651

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掲載種別：研究論文（学術雑誌）  

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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：American Chemical Society (ACS)  

Bioluminescence (BL) generated by luciferase-coelenterazine (CTZ) reactions is broadly employed as an optical readout in bioassays and in vivo molecular imaging. In this study, we demonstrate a systematic approach to elucidate the luciferase-CTZ binding chemistry with a full set of regioisomeric CTZ analogs, where all the functional groups were regiochemically modified. When the chemical structures were categorized into Groups 1–6, the even-numbered Groups (2, 4, and 6) of the CTZ analogs are found to be exceptionally bright with NanoLuc enzyme. A CTZ analogue M2 was the brightest with NanoLuc and the reason was deciphered by a computational analysis of the binding modes. We also report that (i) the regioisomeric CTZ analogs collectively create unique intensity patterns according to each marine luciferase, (ii) the quantitative structure–activity relationship analysis revealed the roles of respective functional groups of CTZ analogs, and (iii) the regioisomeric CTZ analogs also exert red shifts of the BL spectra and color variation: that is, the λmax values are near 500 nm with NanoLuc, near 530 nm with ALuc16, and near 570 nm with RLuc86SG. The advantages of the regioisomeric CTZ analogs were finally demonstrated using (i) a dual-luciferase system with M2-specific NanoLuc and native CTZ-specific ALuc16, (ii) an estrogen activatable single-chain BL probe by imaging, and (iii) BL imaging of live mice bearing tumors expressing NanoLuc and RLuc8.6SG. This study is the first systematic approach to elucidate the regiochemistry in BL imaging studies. This study provides new insights into how CTZ analogs regiochemically work in BL reporter systems and guides the specific applications to molecular imaging.

DOI： 10.1021/acs.bioconjchem.4c00303

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bioluminescence (BL) is an amazing optical readout that has great potential to be utilized in various bioassays and molecular imaging, but also has some unique defects in practical applications. The recent innovative research on BL has enriched the available repertories of the toolbox. While these efforts greatly diversified the users' choices in the applications, the wide choices on the contrary do not promise their successful applications to bioassays. This is mainly due to complexity-driven confusion with the diversity and the lack of accurate knowledge on the advantages and disadvantages of BL. This review is intended to showcase the advantages and disadvantages of BL, and serve as a searchlight to find directions for future studies. We hope that this review provides instant references for readers on BL and leads them to properly understand the "bright" and "dark" sides of BL to narrow down their choices in their applications.

DOI： 10.3389/fchbi.2024.1459397

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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：American Society for Microbiology  

ABSTRACT

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologs of the Ca²⁺/H⁺ antiporter Letm1, a candidate gene for seizures associated with Wolf-Hirschhorn syndrome in humans. Mdm38 is important for K⁺/H⁺ exchange across the inner mitochondrial membrane and contributes to membrane potential formation and mitochondrial protein translation. Ylh47 also localizes to the inner mitochondrial membrane. However, knowledge of the structures and detailed transport activities of Mdm38 and Ylh47 is limited. In this study, we conducted characterization of the ion transport activities and related structural properties of Mdm38 and Ylh47. Growth tests using Na⁺/H⁺ antiporter-deficient Escherichia coli strain TO114 showed that Mdm38 and Ylh47 had Na⁺ efflux activity. Measurement of transport activity across E. coli -inverted membranes showed that Mdm38 and Ylh47 had K⁺/H⁺ , Na⁺/H⁺ , and Li⁺/H⁺ antiport activity, but unlike Letm1, they lacked Ca²⁺/H⁺ antiport activity. Deletion of the ribosome-binding domain resulted in decreased Na⁺ efflux activity in Mdm38. Structural models of Mdm38 and Ylh47 identified a highly conserved glutamic acid in the pore-forming membrane-spanning region. Replacement of this glutamic acid with alanine, a non-polar amino acid, significantly impaired the ability of Mdm38 and Ylh47 to complement the salt sensitivity of E. coli TO114. These findings not only provide important insights into the structure and function of the Letm1-Mdm38-Ylh47 antiporter family but by revealing their distinctive properties also shed light on the physiological roles of these transporters in yeast and animals.

IMPORTANCE

The inner membrane of mitochondria contains numerous ion transporters, including those facilitating H⁺ transport by the electron transport chain and ATP synthase to maintain membrane potential. Letm1 in the inner membrane of mitochondria in animals functions as a Ca²⁺/H⁺ antiporter. However, this study reveals that homologous antiporters in mitochondria of yeast, Mdm38 and Ylh47, do not transport Ca²⁺ but instead are selective for K⁺ and Na⁺ . Additionally, the identification of conserved amino acids crucial for antiporter activity further expanded our understanding of the structure and function of the Letm1-Mdm38-Ylh47 antiporter family.

DOI： 10.1128/jb.00182-24

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掲載種別：研究論文（学術雑誌）  

DOI： 10.5796/denkikagaku.24-fe0016

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Water behavior on protein surfaces influences the protein structure and function. Antifreeze proteins (AFPs) have been intensively studied in the context of biological cytotechnology. AFPs can inhibit the growth of ice microcrystals by forming unique water cluster networks that are influenced by protein surface morphology and hydrophobicity. One such unique water cluster network has been identified as semi-clathrate structures in crystals and is believed to be stabilized by intermolecular interactions within the confined environment. However, there is little atomic-level information about the process of formation of semi-clathrates and the structural units of water-clathrate networks. We identified a single semi-clathrate formed on the pore surface of ferritin crystal, which has a structure similar to that of a natural AFP. Comparison of ferritin mutants and determination of temperature-dependent structures revealed that semi-clathrate water molecules on an α-helix undergo structural alterations with increasing temperatures. Lowering the temperature regenerates the semi-clathrate structure. Water molecules hydrogen-bonded to main chain carbonyl groups are stably immobilized at room temperature and serve as starting points for clathrate formation. These findings provide a mechanistic understanding of water networks in AFPs and guidelines for designing new cryomaterials.

DOI： 10.1021/acs.cgd.3c00880

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

Quinonoid dihydropteridine reductase (QDPR) catalyzes the reduction of quinonoid-form dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4). BH4 metabolism is a drug target for neglected tropical disorders, as trypanosomatid protozoans, including Leishmania and Trypanosoma, require exogenous sources of biopterin for growth. Although QDPR is a key enzyme for maintaining intracellular BH4 levels, the precise catalytic properties and reaction mechanisms of QDPR are poorly understood due to the instability of quinonoid-form substrates. In this study, we analyzed the binding profile of qBH2 to human QDPR in combination with in silico and in vitro methods. First, we performed docking simulation of qBH2 to QDPR to obtain possible binding modes of qBH2 at the active site of QDPR. Then, among them, we determined the most plausible binding mode using molecular dynamics simulations revealing its atomic-level interactions and confirmed it with the in vitro assay of mutant enzymes. Moreover, it was found that not only qBH2 but also quinonoid-form dihydrofolate (qDHF) could be potential physiological substrates for QDPR, suggesting that QDPR may be a bifunctional enzyme. These findings in this study provide important insights into biopterin and folate metabolism and would be useful for developing drugs for neglected tropical diseases.

DOI： 10.1093/jb/mvad062

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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  

In this study, a series of new artificial luciferases (ALucs) was created using sequential insights on missing peptide blocks, which were revealed using the alignment of existing ALuc sequences. Through compensating for the missing peptide blocks in the alignment, 10 sibling sequences were artificially fabricated and named from ALuc55 to ALuc68. The phylogenetic analysis showed that the new ALucs formed an independent branch that was genetically isolated from other natural marine luciferases. The new ALucs successfully survived and luminesced with native coelenterazine (nCTZ) and its analogs in living mammalian cells. The results showed that the bioluminescence (BL) intensities of the ALucs were interestingly proportional to the length of the appended peptide blocks. The computational modeling revealed that the appended peptide blocks created a flexible region near the active site, potentially modulating the enzymatic activities. The new ALucs generated various colors with maximally approximately 90 nm redshifted BL spectra in orange upon reaction with the authors’ previously reported 1- and 2-series coelenterazine analogs. The utilities of the new ALucs in bioassays were demonstrated through the construction of single-chain molecular strain probes and protein fragment complementation assay (PCA) probes. The success of this study can guide new insights into how we can engineer and functionalize marine luciferases to expand the toolbox of optical readouts for bioassays and molecular imaging.

DOI： 10.3390/s23146376

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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  

Albumin assays in serum are important for the prognostic assessment of many life-threatening diseases, such as heart failure, liver disease, malnutrition, inflammatory bowel disease, infections, and kidney disease. In this study, synthetic coelenterazine (CTZ) indicators are developed to quantitatively illuminate human and bovine serum albumins (HSA and BSA) with high specificity. Their functional groups were chemically modified to specifically emit luminescence with HSA and BSA. The CTZ indicators were characterized by assaying the most abundant serum proteins and found that the CTZ indicators S6 and S6h were highly specific to HSA and BSA, respectively. Their colors were dramatically converted from blue, peaked at 480 nm, to yellowish green, peaked at 535 nm, according to the HSA–BSA mixing ratios, wherein the origins and mixing levels of the albumins can be easily determined by their colors and peak positions. The kinetic properties of HSA and BSA were investigated in detail, confirming that the serum albumins catalyze the CTZ indicators, which act as pseudo-luciferases. The catalytic reactions were efficiently inhibited by specific inhibitors, blocking the drug-binding sites I and II of HSA and BSA. Finally, the utility of the CTZ indicators was demonstrated through a quantitative imaging of the real fetal bovine serum (FBS). This study is the first example to show that the CTZ indicators specify HSA and BSA with different colors. This study contributes to the expansion of the toolbox of optical indicators, which efficiently assays serum proteins in physiological samples. Considering that these CTZ indicators immediately report quantitative optical signals with high specificity, they provide solutions to conventional technical hurdles on point-of-care assays of serum albumins.

DOI： 10.3390/s23136020

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

多細胞動物のからだの形作りは，隣り合う細胞と細胞をつなぐカドヘリン間の結合によって制御されているが，その結合機構については不明な点が多かった．高速原子間力顕微鏡を用いて，溶液中におけるカドヘリンの結合過程を一分子のスケールで直接可視化することで，カドヘリンの結合機構を解析した．

DOI： 10.2142/biophys.63.104

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掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Imaging protein–protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.

DOI： 10.3390/s23073498

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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  

Phototrophic bacteria face diurnal variations of environmental conditions such as light and osmolarity, that affect their carbon metabolism and ability to generate organic compounds. The model cyanobacterium, Synechocystis sp. PCC 6803 forms a biofilm when it encounters extreme conditions like high salt stress, but the molecular mechanisms involved in perception of environmental changes that lead to biofilm formation are unknown. Here, we studied two two-component regulatory systems (TCSs) that contain diguanylate cyclases (DGCs), which produce the second messenger c-di-GMP, as potential components of the biofilm-inducing signaling pathway in Synechocystis. Analysis of single mutants provided evidence for involvement of Rre2 and Rre8 in biofilm formation. A bacterial two-hybrid assay showed that the response regulators, Rre2 and Rre8 each formed a TCS with a specific histidine kinase, Hik12 and Hik14, respectively. The in vitro assay showed that Rre2 had DGC activity regardless of its de/phosphorylation status, whereas Rre8 required phosphorylation for DGC activity. Hik14-Rre8 likely functioned as an inducible sensing system in response to environmental change. Biofilm assays with Synechocystis mutants suggested that pairs of hik12-rre2 and hik14-rre8 responded to high salinity-induced biofilm formation. Inactivation of hik12-rre2 and hik14-rre8 did not affect the performance of the light reactions of photosynthesis. These data suggest that Hik12-Rre2 and Hik14-Rre8 participate in biofilm formation in Synechocystis by regulating c-di-GMP production via the DGC activity of Rre2 and Rre8.

DOI： 10.1111/mmi.15057

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Bioluminescence (BL) is broadly used as an optical readout in bioassays and molecular imaging. In this study, the near-infrared (NIR) BL imaging systems were developed. The system was harnessed by prototype copepod luciferases, artificial luciferase 30 (ALuc30) and its miniaturized version picALuc, and were characterized with 17 kinds of coelenterazine (CTZ) analogues carrying bulky functional groups or cyanine 5 (Cy5). They were analyzed of BL spectral peaks and enzymatic kinetics, and explained with computational modeling. The results showed that (1) the picALuc-based system surprisingly boosts the BL intensities predominantly in the red and NIR region with its specific CTZ analogues; (2) both ALuc30- and picALuc-based systems develop unique through-bond energy transfer (TBET)-driven spectral bands in the NIR region with a Cy5-conjugated CTZ analogue (Cy5-CTZ); and (3) according to the computational modeling, the miniaturized version, picALuc, has a large binding pocket, which can accommodate CTZ analogues containing bulky functional groups and thus allowing NIR BL. This study is an important addition to the BL imaging toolbox with respect to the development of orthogonal NIR reporter systems applicable to physiological samples, together with the understanding of the BL-emitting chemistry of marine luciferases.

DOI： 10.1007/s43630-023-00367-8

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その他リンク： https://link.springer.com/article/10.1007/s43630-023-00367-8/fulltext.html

掲載種別：研究論文（学術雑誌）   出版者・発行元：Royal Society of Chemistry (RSC)  

Protein crystals can be utilized as porous scaffolds to capture exogenous molecules. Immobilization of target proteins using protein crystals is expected to facilitate X-ray structure analysis of proteins that are difficult to be crystallized. One of the advantages of scaffold-assisted structure determination is the analysis of metastable structures that are not observed in solution. However, efforts to fix target proteins within the pores of scaffold protein crystals have been limited due to the lack of strategies to control protein–protein interactions formed in the crystals. In this study, we analyze the metastable structure of the miniprotein, CLN025, which forms a β-hairpin structure in solution, using a polyhedra crystal (PhC), an in-cell protein crystal. CLN025 is successfully fixed within the PhC scaffold by replacing the original loop region. X-ray crystal structure analysis and molecular dynamics (MD) simulation reveal that CLN025 is fixed as a helical structure in a metastable state by non-covalent interactions in the scaffold crystal. These results indicate that modulation of intermolecular interactions can trap various protein conformations in the engineered PhC and provides a new strategy for scaffold-assisted structure determination.

DOI： 10.1039/d2bm01759h

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掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

A unique combinatorial bioluminescence (BL) imaging system was developed for determining molecular events in mammalian cells with various colors and BL intensity patterns. This imaging system consists of one or multiple reporter luciferases and a series of novel coelenterazine (CTZ) analogues named “S-series”. For this study, ten kinds of novel S-series CTZ analogues were synthesized and characterized concerning the BL intensities, spectra, colors, and specificity of various marine luciferases. The characterization revealed that the S-series CTZ analogues luminesce with blue-to-orange-colored BL spectra with marine luciferases, where the most red-shifted BL spectrum peaked at 583 nm. The colors completed a visible light color palette with those of our precedent C-series CTZ analogues. The synthesized substrates S1, S5, S6, and S7 were found to have a unique specificity with marine luciferases, such as R86SG, NanoLuc (shortly, NLuc), and ALuc16. They collectively showed unique BL intensity patterns to identify the marine luciferases together with colors. The marine luciferases, R86SG, NLuc, and ALuc16, were multiplexed into multi-reporter systems, the signals of which were quantitatively unmixed with the specific substrates. When the utility was applied to a single-chain molecular strain probe, the imaging system simultaneously reported three different optical indexes for a ligand, i.e., unique BL intensity and color patterns for identifying the reporters, together with the ligand-specific fold intensities in mammalian cells. This study directs a new combinatorial BL imaging system to specific image molecular events in mammalian cells with multiple optical indexes.

DOI： 10.3390/ijms24021420

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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  

The present study introduces a unique BL signature imaging system with novel CTZ analogues named “C-series.” Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that the four CTZ analogues named C3, C4, C6, and C7, individually or collectively luminesce with completely distinctive BL spectral signatures and intensity patterns according to the luciferases: Renilla luciferase (RLuc), NanoLuc, and artificial luciferase (ALuc). The signatural reporters were multiplexed into a multi-reporter system comprising RLuc8.6-535SG and ALuc16. The usefulness of the signatural reporters was further determined with a multi-probe system that consists of two single-chain probes embedding RLuc8 and ALuc23. This study is a great addition to the study of conventional bioassays with a unique methodology, and for the specification of each signal in a single- or multi-reporter system using unique BL signatures and patterns of reporter luciferases.

DOI： 10.3390/ijms232113047

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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：The Physiological Society  

Boric acid is a vital micronutrient that is toxic at high concentrations in animals. However, the mechanisms underlying boric acid transport in animal cells remain unclear. To identify the plasma membrane boric acid channels in animals, we analyzed the function of human aquaporins (AQPs), which are homologous to the nodulin-like intrinsic protein family of plant boric acid channels. When human AQPs were expressed in Xenopus laevis oocytes, the results of the swelling assay showed that boric acid permeability significantly increased in oocytes expressing AQP3, 7, 8, 9, and 10, but not in those expressing AQP1, 2, 4, and 5. The boric acid influxes of these oocytes were also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these AQPs transported boric acid (B(OH)3 ) but not borate ions (B(OH)4 - ). These results indicate that AQP3, 7, 8, 9, and 10 act as boric acid transport systems, likely as channels in humans.

DOI： 10.14814/phy2.15164

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.14814/phy2.15164

掲載種別：研究論文（学術雑誌）   出版者・発行元：The Japanese Society of Applied Glycoscience  

Cellobiohydrolase I from Trichoderma reesei (TrCel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of TrCel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of TrCel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of TrCel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of TrCel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake (kon for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.

DOI： 10.5458/jag.jag.jag-2020_0014

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The present study indicated that the mixed lipid bilayer of dimyristoylphosphatidylcholine (DMPC) and trehalosemonomyristate (TreC14) interacted strongly with the plasma membrane of cancer cells, and not that of normal cells, when the composition of TreC14 was 70%, as revealed by coarse-grained molecular dynamics simulations. These results were consistent with those of previous experimental studies, indicating that DMPC/TreC14 mixed liposomes (DMTreC14) with TreC14 composition at 70% exhibited a strong anti-cancer effect without affecting normal cells. The simulations also revealed that lipids with highly hydrophilic and bulky head groups, such as TreC14, phosphatidylinositol (PI), and phosphatidylserine (PS), showed the tendency to accumulate. This caused both the DMTreC14 and cancer cell membranes to bend into large positive curvatures, resulting in tight contact between them. In contrast, no apparent interaction between the DMTreC14 and normal cell membranes was observed because PI and PS did not exist in the extracellular monolayer of the normal cell membrane.

DOI： 10.1016/j.bbrep.2021.100913

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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：Informa UK Limited  

Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K+ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.

DOI： 10.1080/19336950.2020.1825894

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Royal Society of Chemistry (RSC)  

The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.

DOI： 10.1039/d0cp02069a

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Microbiology Society  

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger known to control a variety of bacterial processes. The model cyanobacterium, Synechocystis sp. PCC 6803, has a score of genes encoding putative enzymes for c-di-GMP synthesis and degradation. However, most of them have not been functionally characterized. Here, we chose four genes in Synechocystis (dgcA-dgcD), which encode proteins with a GGDEF, diguanylate cyclase (DGC) catalytic domain and multiple Per-ARNT-Sim (PAS) conserved regulatory motifs, for detailed analysis. Purified DgcA, DgcB and DgcC were able to catalyze synthesis of c-di-GMP from two GTPs in vitro. DgcA had the highest activity, compared with DgcB and DgcC. DgcD did not show detectable activity. DgcA activity was specific for GTP and stimulated by the divalent cations, magnesium or manganese. Full activity of DgcA required the presence of the multiple PAS domains, probably because of their role in protein dimerization or stability. Synechocystis mutants carrying single deletions of dgcA-dgcD were not affected in their growth rate or biofilm production during salt stress, suggesting that there was functional redundancy in vivo. In contrast, overexpression of dgcA resulted in increased biofilm formation in the absence of salt stress. In this study, we characterize the enzymatic and physiological function of DgcA-DgcD, and propose that the PAS domains in DgcA function in maintaining the enzyme in its active form.

DOI： 10.1099/mic.0.000929

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society of Japan  

Motor proteins are essential units of life and are well-designed nanomachines working under thermal fluctuations. These proteins control moving direction by consuming chemical energy or by dissipating electrochemical potentials. Chitinase A from bacterium Serratia marcescens (SmChiA) processively moves along crystalline chitin by hydrolysis of a single polymer chain to soluble chitobiose. Recently, we directly observed the stepping motions of SmChiA labeled with a gold nanoparticle by dark-field scattering imaging to investigate the moving mechanism. Time constants analysis revealed that SmChiA moves back and forth along the chain freely, because forward and backward states have a similar free energy level. The similar probabilities of forward-step events (83.5%=69.3%+14.2%) from distributions of step sizes and chain-hydrolysis (86.3%=(1/2.9)/(1/2.9+1/18.3)×100) calculated from the ratios of time constants of hydrolysis and the backward step indicated that SmChiA moves forward as a result of shortening of the chain by a chitobiose unit, which stabilizes the backward state. Furthermore, X-ray crystal structures of sliding intermediate and molecular dynamics simulations showed that SmChiA slides forward and backward under thermal fluctuation without large conformational changes of the protein. Our results demonstrate that SmChiA is a burnt-bridge Brownian ratchet motor.

DOI： 10.2142/biophysico.bsj-2020004

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セラチア菌由来キチン加水分解酵素SmChiAは，結晶性キチン上を運動する様子が初めて報告されたキチナーゼの一つである．しかしその運動機構は未だ不明であった．そこでSmChiAを金属ナノ粒子で標識し，全反射暗視野顕微鏡を用いてその運動を高速高精度で計測することにより運動機構の解明を試みた．その結果，SmChiAは主にジアセチルキトビオース単位に相当する1.1 nm刻みで前後に運動しており，その前進運動の確率は後退運動に対する加水分解反応の割合とほぼ同じであることがわかった．よってSmChiAは熱ゆらぎにより前後に運動し，分子鎖末端からキトビオースを切り離すことにより後退運動を抑制して運動方向性を生み出す“burnt-bridge”ブラウニアンラチェット酵素であると推定された．

DOI： 10.5458/bag.10.2_89

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.

DOI： 10.1039/D0NR01121E

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We investigated experimentally whether a short peptide, called PvLEA-22, which consists of two tandem repeats of an 11-mer motif of Group 3 late embryogenesis abundant proteins, has a chaperone-like function for denatured proteins. Lysozyme was selected as a target protein. Turbidity measurements indicated that the peptide suppresses the heat-induced aggregation of lysozyme when added at a molar ratio of PvLEA-22/lysozyme > 40. CD and DSC measurements confirmed that the lysozyme was denatured on heating, but spontaneously refolded on subsequent cooling in the presence of the peptide. As a result, up to 80% of the native catalytic activity of lysozyme was preserved. Similar chaperone-like activity was also observed for a peptide with the same amino acid composition as PvLEA-22, but whose sequence is scrambled. To elucidate the underlying mechanism of the chaperone function of these peptides, we performed coarse-grained molecular dynamics simulations. This revealed that a denatured lysozyme molecule is shielded by several peptide molecules in aqueous solution, which act as a physical barrier, reducing the opportunities for collision between denatured proteins. An important finding was that a peptide bound to the denatured protein is very rapidly replaced by another: due to such rapid exchange, peptide-protein contact time is very short, on the order of ~200 ns. Therefore, the peptide does not constrain the behavior of the denatured protein, which can refold freely.

DOI： 10.1021/acs.jpcb.9b11000

PubMed

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Substrate-assisted catalysis (SAC), a mechanism of chitin hydrolysis by chitinases belonging to the glycoside hydrolase family 18 (GH18), has been studied experimentally and theoretically for several decades. However, the detailed reaction mechanism in chitinase A (ChiA) remains unclear at the atomic level. In this study, we investigated glycosylation, the first step of SAC, of ChiA obtained from Serratia marcescens (SmChiA), using QM/MM simulations combined with a one-dimensional projection (ODP) technique, which enabled us to explore the multi-dimensional free energy surface efficiently. The results showed that the reaction proceeds via a novel ring-shaped concerted reaction pathway with interconvertible intermediates, viz. oxazolinium ion and oxazoline, which have not been fully identified in previous studies. We also compared this chitin hydrolysis mechanism in SmChiA with that in SmChiB reported previously. The computational protocol developed in this study could also be applicable for elucidating complicated reaction mechanisms in other enzymes.

DOI： 10.1039/C9CP05163E

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.2142/biophys.59.330

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Group 3 late embryogenesis abundant (G3LEA) proteins, which act as a well-characterized desiccation protectant in anhydrobiotic organisms, are structurally disordered in solution, but they acquire a predominantly α-helical structure during drying. Thus, G3LEA proteins are now accepted as intrinsically disordered proteins (IDPs). Their functional regions involve characteristic 11-mer repeating motifs. In the present study, to elucidate the origin of the IDP property of G3LEA proteins, we applied replica exchange molecular dynamics (REMD) simulation to a model peptide composed of two tandem repeats of an 11-mer motif and its counterpart peptide whose amino acid sequence was randomized with the same amino acid composition as that of the 11-mer motif. REMD simulations were performed for a single α-helical chain of each peptide and its double-bundled strand in a wide water content ranging from 5 to 78.3 wt%. In the latter case, we tested different types of arrangement: 1) the dipole moments of the two helices were parallel or anti-parallel and 2) due to the amphiphilic nature of the α-helix of the 11-mer motif, two types of the side-to-side contact were tested: hydrophilic-hydrophilic facing or hydrophobic-hydrophobic facing. Here, we revealed that the single chain alone exhibits no IDP-like properties, even if it involves the 11-mer motif, and the hydrophilic interaction of the two chains leads to the formation of a left-handed α-helical coiled coil in the dry state. These results support the cytoskeleton hypothesis that has been proposed as a mechanism by which G3LEA proteins work as a desiccation protectant.

DOI： 10.2142/biophysico.16.0_196

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that belongs to the ATP binding cassette protein superfamily. Deletion of phenylalanine at position 508 (ΔF508) is the most common CF-associated mutation and is present in nearly 90% of CF patients. Currently, atomistic level studies are insufficient for understanding the mechanism by which the deletion of a single amino acid causes greatly reduced folding as well as trafficking and gating defects. To clarify this mechanism, we first constructed an atomic model of the inward-facing ΔF508-CFTR and performed all-atom molecular dynamics (MD) simulations of the protein in a membrane environment. All of the computational methodologies used are based on those developed in our previous study for wild-type CFTR. Two important findings were obtained. First, consistent with several previous computational results, the deletion of F508 causes a disruption of a hydrophobic cluster located at the interface between the nucleotide binding domain 1 (NBD1) and intracellular loop 4 (ICL4). This exerts unfavorable influences on the correlated motion between ICLs and transmembrane domains (TMDs), likely resulting in gating defects. Second, the F508 deletion affected the NBD1–NBD2 interface via allosteric communication originating from the correlated motion between NBDs and ICLs. As a result, several unusual inter-residue interactions are caused at the NBD1–NBD2 interface. In other words, their correct dimerization is impaired. This study provided insight into the atomic-level details of structural and dynamics changes caused by the ΔF508 mutation and thus provides good insight for drug design.

DOI： 10.2142/biophysico.15.0_33

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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 and Sons Inc.  

DOI： 10.1002/prot.25433

Scopus

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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：一般社団法人 日本生物物理学会  

<p>Upon the binding of ATP molecules to nucleotide binding domains (NBDs), ATP-binding cassette (ABC) exporters undergo a conformational transition from an inward-facing (IF) to an outward-facing (OF) state. This molecular event is a typical example of chemo-mechanical coupling. However, the underlying mechanism remains unclear. In this study, we analyzed the IF→OF transition of a representative ABC exporter, MsbA, by solving the equation of motion under an elastic network model (ENM). ATP was represented as a single node in ENM or replaced by external forces. When two ATP nodes were added to the ENM of the IF state protein, the two NBDs dimerized; subsequently, the two transmembrane domains opened toward the extracellular side, resulting in the formation of the OF structure. Such a conformational transition was also reproduced by applying external forces, which caused the rotational motion of the NBDs instead of the addition of ATP nodes. The process of the conformational transition was analyzed in detail using cross-correlation maps for node-node interactions. More importantly, it was revealed that the ATP binding energy is converted into distortion energy of several transmembrane helices. These results are useful for understanding the chemo-mechanical coupling in ABC transporters.</p>

DOI： 10.2142/biophysico.14.0_161

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.20585/cryobolcryotechnol.63.2_119

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1042/BCJ20161051

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1246/bcsj.20160261

Web of Science

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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/acs.jpcb.6b07332

Web of Science

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：低温生物工学会  

DOI： 10.20585/cryobolcryotechnol.62.2_133

CiNii Books

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：低温生物工学会  

DOI： 10.20585/cryobolcryotechnol.62.2_127

CiNii Books

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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/acs.jpcb.6b05611

Web of Science

J-GLOBAL

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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.cplett.2015.11.014

Web of Science

J-GLOBAL

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担当区分：責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：一般社団法人 日本生物物理学会  

ATP-binding cassette (ABC) transporters constitute one of the largest superfamilies of membrane proteins that translocate variable substrates through the transmembrane domains (TMDs) powered by the ATP-driven nucleotide-binding domain (NBDs) engines. The coupling helices (CHs) located at the NBD-TMD interfaces play important roles in the structural transition between the inward- and outward-facing conformations. Here, we review our recent studies on the roles of CHs and the effects of ATP and substrates in the functional dynamics of ABC transporters.

DOI： 10.2142/biophys.56.005

CiNii Books

J-GLOBAL

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その他リンク： http://search.jamas.or.jp/link/ui/2016206967

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：低温生物工学会  

Group 3 late embryogenesis abundant (G3LEA) proteins are capable of protecting liposomes from desiccation damage. In our previous studies, a model peptide (PvLEA-22) which has two tandem repeats of the characteristic 11-mer motif found in G3LEA proteins was shown to have a similar protective function for dried liposomes. However, its underlying mechanism is still unclear. It has been hypothesized that PvLEA-22 could prevent direct membrane-membrane contacts by shielding the lipid membrane surface. In this study, to confirm the validity of this hypothesis, we performed molecular dynamics (MD) simulation combined with the so-called umbrella sampling method for a model system composed of PvLEA-22 and a membrane bilayer, and analyzed the free energy profile for the binding process of the peptide onto the membrane surface, and additionally the structure of the bound peptide. As a result, it was shown that the peptide is able to bind to the membrane with a relatively large binding free energy of 18 kcal/mol through a barrierless process, and it has a propensity to form a β-sheet-like structure on the membrane surface due to its characteristic sequence.

DOI： 10.20585/cryobolcryotechnol.61.2_105

CiNii Books

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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/acs.jpcb.5b05432

Web of Science

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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/jp507930e

Web of Science

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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.cplett.2014.10.038

Web of Science

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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.febslet.2014.09.004

Web of Science

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：低温生物工学会  

We here report the results of MD simulations for several systems including a model peptide which has two tandem repeats of the characteristic 11-mer motif found in group 3 late embryogenesis abundant (G3LEA) proteins and its partner protein (lysozyme and HybD) in aqueous solution. And for comparison, we performed MD simulations for a control peptide which has the same amino acid composition as that of the G3LEA model but a scrambled sequence. As a result, the G3LEA model was found to spontaneously approach its partner and form the association complex. According to detailed analysis, the acidic residues (Glu and Asp) and basic residues (Lys) in the G3LEA model contribute to binding to lysozyme and HybD, respectively. Similar results were obtained for the control peptide.

DOI： 10.20585/cryobolcryotechnol.60.2_89

CiNii Books

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131-L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.

DOI： 10.1371/journal.pone.0103397

PubMed

J-GLOBAL

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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：Chemical Society of Japan  

DOI： 10.1246/bcsj.20140136

Scopus

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c4ob00673a

Web of Science

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：低温生物工学会  

We show that a short model peptide which has two tandem repeats of the 11-mer motif of a group3 late embryogenesis abundant protein protects proteins against aggregation by desiccation stress. Next, we measure the dissociation constant between the LEA model peptide and its partner protein (lysozyme and HybD) using quartz crystal microbalance (QCM) method. Similar experiments are performed for a native LEA protein from the anhydrobiotic nematode A. avenae. On the basis of these results, the mechanism of the anti-aggregation function of the LEA peptide is discussed.

DOI： 10.20585/cryobolcryotechnol.59.2_95

CiNii Books

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：低温生物工学会  

Here, we have performed molecular dynamics (MD) simulations of a short model peptide which has two tandem repeats of the 11-mer motif of a group3 late embryogenesis abundant protein in aqueous solution. First we showed that this peptide (LEA peptide) adopts turn-like structures in water with the hydrophilic side chains being exposed to the medium. Next, we performed MD simulations for the mixed system of the LEA peptide and lysozyme and showed that the LEA peptide binds to the surface of lysozyme through the intermolecular hydrogen bonds with three different binding patterns. Interestingly, the conformation of the LEA peptide is transformed into more extended form with binding to lysozyme. It is concluded that the LEA peptide could protect lysozyme from aggregation by shielding the protein surface in the dry state. In other words, the LEA peptide works as a chemical chaperon.

DOI： 10.20585/cryobolcryotechnol.59.2_101

CiNii Books

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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.cplett.2012.12.040

Web of Science

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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/jp308315w

Web of Science

PubMed

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記述言語：日本語   出版者・発行元：日本医科大学医学会  

We review our recent collaborative study, performed by computational physicists and biochemists, of the enzyme effects due to the drug called febuxostat. Febuxostat, which was recently approved in the US, European Union and Japan for treatment of gout, inhibits xanthine oxidoreductase (XOR)-mediated generation of uric acid during purine catabolism. Experiments have shown that febuxostat has strong effects on mammalian XOR but not on bacterial XOR, although the two enzymes have similar three-dimensional structures. To clarify the difference in the inhibitory power of febuxostat, we performed docking and molecular dynamics simulations for mammalian and bacterial XORs. We found that the static structures are not sufficient to explain the binding difference and that important interactions occur between febuxostat and the active region of the enzymes which suggests a better strategy for drug design.

DOI： 10.1272/manms.8.222

CiNii Books

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その他リンク： http://t2r2.star.titech.ac.jp/cgi-bin/publicationinfo.cgi?q_publication_content_number=CTT100641427

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pcbi.1002555

Web of Science

J-GLOBAL

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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.cplett.2009.03.009

Scopus

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

In this paper, we introduce an effective kinetic model for the linear polymerization of cytoskeletal filaments with a threshold length in a finite-volume system in order to show the importance of a threshold length in the nucleation phase and to understand the kinetic constant accurately. We analyze this model numerically for several concentrations and found some kind of shifted peak in the length distribution of linear polymers that were longer than the threshold length. We also found that the average length of filaments in this model depended on the threshold length exponentially and that the peaks relaxed with time. These suggest that nucleation is a critical factor in cellular phenomena.

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記述言語：日本語   掲載種別：研究論文（研究会，シンポジウム資料等）  

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その他リンク： http://hdl.handle.net/2433/97679

担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

We analyze growth–shrinkage dynamics of cytoskeletons by using a lattice model in two dimensions. The behavior of the length of a chain corresponding to one cytoskeleton immersed in a “gas” of the basic units of cytoskeletons is simulated for low and intermediate concentrations, which shows the characteristic behavior of dynamic instability. We calculate the mean long-time velocity for several low concentrations and find that the length dynamics is sensitive to the concentration, which suggests the existence of a critical concentration. We also calculate the mean velocity for several short time spans and the probability distribution functions (PDFs) of the length difference with different time lags Δt. We find that the PDFs have a shoulder at around zero and a peak in the positive velocity region and the central parts of the PDF are essentially independent of Δt.

DOI： 10.1016/S0378-4371(02)01064-6

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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）  

DOI： 10.1063/1.1291613

Web of Science

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[Tokyo Tech] https://www.titech.ac.jp/news/2024/069338
[Tokyo Tech bio] https://educ.titech.ac.jp/bio/news/2024_06/066173.html
[RIKEN] https://www.riken.jp/press/2024/20240614_1/index.html
[EurekAlert!] https://www.eurekalert.org/news-releases/1048185
[Phys.org] https://shorturl.at/3M4Ux (New method enables fast crystal structure analysis of intrinsically disordered proteins)

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[JAIST] https://www.jaist.ac.jp/whatsnew/press/2024/05/31-1.html
[Tokyo Tech] https://www.titech.ac.jp/news/2024/069365
[Tokyo Tech bio] https://educ.titech.ac.jp/bio/news/2024_05/066110.html
[EurekAlert!] https://www.eurekalert.org/news-releases/1046470
[AlphaGalileo] https://www.alphagalileo.org/en-gb/Item-Display/ItemId/246481

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[Tokyo Tech] https://www.titech.ac.jp/news/2023/068060 (X) https://cutt.ly/jwHccWrN
[Tokyo Tech bio] https://educ.titech.ac.jp/bio/news/2023_12/065211.html
[Kindai] https://newscast.jp/news/3351241
[EurekAlert!] https://cutt.ly/JwHcx3Tr

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[ExCELLS] https://www.excells.orion.ac.jp/news/5597 (twitter) https://cutt.ly/7LL8SqO
[Nagoya Univ] https://www.nagoya-u.ac.jp/researchinfo/result/2022/07/post-289.html (twitter) https://cutt.ly/sLVOEvq
[Tokyo Tech] https://www.titech.ac.jp/news/2022/064498 (twitter) https://cutt.ly/0LL3Lbt
[Tokyo Tech bio] https://educ.titech.ac.jp/bio/news/2022_07/062898.html
[EurekAlert!] https://cutt.ly/1LL7qHF
[Bioengineer.org] https://cutt.ly/jLL7tOK
[Phys.org] https://cutt.ly/MLL47pp

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[Tokyo Tech] https://www.titech.ac.jp/news/2022/063746
(twitter) https://cutt.ly/QGjC3xb
[Tokyo Tech bio (5/12)] https://educ.titech.ac.jp/bio/news/2022_05/062469.html

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[jiji (社会)] https://sp.m.jiji.com/article/show/2743091 (https://cutt.ly/rGzMnRh) (twitter) https://cutt.ly/QGz1AJv (時事メディカル) https://cutt.ly/fGz1Wtj (twitter) https://cutt.ly/6Gz1D03
同 Yahoo!ニュース (https://cutt.ly/kGzMM1d), Yahoo!ファイナンス (https://cutt.ly/uGz0NA4), exciteニュース (https://cutt.ly/UGz1rh8), gooニュース (https://cutt.ly/RGz17rU), BIGLOBEニュース (https://cutt.ly/KGz0rwH), dmenuニュース (https://cutt.ly/UGz0gy2), au Webポータルニュース (https://cutt.ly/RGz0bef), @niftyニュース (https://cutt.ly/LGz3L5c), msnニュース (https://cutt.ly/oGbz0RY)
[jiji (企業)] 東工大・島津、世界最小級の発光酵素開発 https://sp.m.jiji.com/article/show/2743197
同 Yahoo!ニュース (https://cutt.ly/rGzMI0D), gooニュース (https://cutt.ly/9Gz13kT), dmenuニュース (https://cutt.ly/KGz0aRK)

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[nikkei] https://www.nikkei.com/nkd/company/article/?&ng=DGXZRSP631231_V20C22A4000000&scode=7701
(article) https://cutt.ly/cGSZJoW

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[nikkeibp] https://bio.nikkeibp.co.jp/atcl/news/p1/22/04/25/09417/

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[SHIMADZU] https://www.shimadzu.co.jp/news/press/3dzmjf2p8wyq6cw5.html
(twitter) https://cutt.ly/mGjCZgo

「picALuc」サンプル問い合わせフォーム⇒ https://solutions.shimadzu.co.jp/form/press/picaluc/contact.html

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[Tokyo Tech] https://www.titech.ac.jp/news/2022/062778
[Tokyo Tech bio] https://educ.titech.ac.jp/bio/news/2022_01/061773.html
[Kyushu Univ] https://www.kyushu-u.ac.jp/ja/researches/view/712/
[Nagoya Univ] https://www.nagoya-u.ac.jp/about-nu/public-relations/researchinfo/upload_images/20220107_sci.pdf
[ExCELLS] https://www.excells.orion.ac.jp/news/4758
[EurekAlert!] https://cutt.ly/hGjBBde
[Bioengineer.org] https://cutt.ly/1GjBZha
[Phys.org] https://cutt.ly/dGjBGlZ
[ScienceDaily] https://cutt.ly/MGjBA32

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[Tokyo Tech] https://www.titech.ac.jp/news/2019/045898.html
[Tokyo Tech bio] https://educ.titech.ac.jp/bio/news/2019_12/058335.html
[EurekAlert!] https://cutt.ly/lGjBmsp
[Phys.org] https://cutt.ly/sGjBYtw

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[IMS] https://www.ims.ac.jp/news/2018/09/19_4057.html
[Tokyo Tech] https://www.titech.ac.jp/news/2018/042389.html
[Tokyo Tech bio] https://educ.titech.ac.jp/bio/news/2018_09/056304.html
[EurekAlert!] https://cutt.ly/kGjVZkw
[Phys.org] https://cutt.ly/3GjVVzl

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[URL] http://www.a.u-tokyo.ac.jp/topics/2013/20130510-2.html

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[URL] http://www.natureasia.com/ja-jp/srep/abstracts/35132

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[Science Tokyo bio] https://educ.titech.ac.jp/bio/news/2025_08/068204.html

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[mynavi] https://news.mynavi.jp/techplus/article/20220720-2403053/ (twitter) https://cutt.ly/UL1DYey
同 Rakuten Infoseek News (https://cutt.ly/aL1F48W), BIGLOBEニュース (https://cutt.ly/wL1DCDA), exciteニュース (https://cutt.ly/NL1DM4c), ニコニコニュース (https://cutt.ly/kL1FmBi), Mapionニュース (https://cutt.ly/qL1FI20)

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[dempa-digital] https://dempa-digital.com/article/335025 (twitter) https://cutt.ly/oLVVnhd

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[mynavi] https://news.mynavi.jp/techplus/article/20220425-2329591/
(twitter) https://cutt.ly/hGjC6lO
同 Rakuten Infoseek News (https://cutt.ly/cGjVoyL), BIGLOBEニュース (https://cutt.ly/RGjVd1y), exciteニュース (https://cutt.ly/oGjVjN0), gooニュース (https://cutt.ly/PGjVvGG), dmenuニュース (https://cutt.ly/8GjVQnw)

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[dempa-digital] https://dempa-digital.com/article/308708
(twitter) https://cutt.ly/mGj51WZ

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[kyoto-np] https://www.kyoto-np.co.jp/articles/-/778323
(twitter) https://cutt.ly/bGjNx6S
同 Yahoo!ニュース (https://cutt.ly/4GjNbhA), Rakuten Infoseek News (https://cutt.ly/zGjNmYV), exciteニュース(https://cutt.ly/3GjNoVr), gooニュース (https://cutt.ly/9GjNuP2), dmenuニュース (https://cutt.ly/DGjNyiS), au Webポータルニュース (https://cutt.ly/fGjNrDo), 47NEWS (https://cutt.ly/7GjB8xN)

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(オンライン) 島津と東工大、世界最小クラスの発光酵素開発 来年めど製品化
[nikkan] https://www.nikkan.co.jp/articles/view/634827
(ニュースイッチ) https://newswitch.jp/p/31914

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飯野翼くん（現、名大院）の論文が2019 PCCP HOT articleに選ばれました。
DOI: 10.1039/C9CP05163E
[2019 PCCP HOT articles] https://pubs.rsc.org/en/journals/articlecollectionlanding?sercode=cp&themeid=82edaade-4192-47df-9db1-8043135f9b70

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[IMSサイト] https://groups.ims.ac.jp/organization/iino_g/pdf/181005kagakushinbun_NakamuraSmChiA.pdf
(twitter) https://cutt.ly/1GjBdji

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[URL] https://news.mynavi.jp/article/20130513-a238/

同、livedoor NEWS (http://bit.ly/17Sr9uj) 、exciteニュース、夕刊アメーバニュース、Yahoo!ニュース、ニコニコニュース、gooニュース、BIGLOBEニュースなど

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edited by Helmut Grubmüller and Klaus Schulten
in Journal of Structural Biology volume 157, issue 3.
[URL] https://www.sciencedirect.com/journal/journal-of-structural-biology/vol/157/issue/3

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