業績情報

• A macroscopically homogeneous lipid phase exhibits leaflet-specific lipid diffusion in a glass-supported lipid bilayer
  Takuhiro Otosu; Miyuki Sakaguchi; Shoichi Yamaguchi
  Physical Chemistry Chemical Physics, 巻：27, 号：9, 開始ページ：4944, 終了ページ：4949, 2025年02月, ［査読有り］, ［筆頭著者, 責任著者］
  Leaflet-specific lipid diffusion analysis revealed that the strength of interleaflet coupling is sensitive to the lipid composition even in the region where macroscopically homogeneous phase is observed.
  Royal Society of Chemistry (RSC), 研究論文（学術雑誌）
  DOI：https://doi.org/10.1039/d5cp00203f
  DOI ID：10.1039/d5cp00203f, ISSN：1463-9076, eISSN：1463-9084
• A new rigid non-polarizable model for semi-heavy water: TIP4P/2005-SHW
  Tetsuyuki Takayama; Takuhiro Otosu; Shoichi Yamaguchi
  The Journal of Chemical Physics, 巻：161, 号：23, 2024年12月, ［査読有り］
  In molecular dynamics (MD) simulations of water, rigid non-polarizable models are still valuable and widely used because of their low computational cost and excellent performance. Most of such models have been for light water (H2O) for a long time, and a few have recently been reported for heavy water (D2O). The specific models of D2O are needed because of its notably slower dynamics and stronger hydrogen bond than H2O. To our knowledge, no models of semi-heavy water (HOD) were made, although HOD is the most abundant and, therefore, most important chemical species in isotopically diluted water (IDW) prepared by mixing equal amounts of H2O and D2O. For precise MD simulations of IDW, a specific model of HOD is definitely needed. Here, we report the development of a new rigid non-polarizable model of HOD, TIP4P/2005-SHW, on the basis of the most popular H2O model, TIP4P/2005, and its D2O counterpart, TIP4P/2005-HW. We show the details of the development and demonstrate the high reproducibility of TIP4P/2005-SHW in terms of the density, the temperature of maximum density, the viscosity, and the diffusion coefficient.
  AIP Publishing, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1063/5.0240271
  DOI ID：10.1063/5.0240271, ISSN：0021-9606, eISSN：1089-7690
• Diffusion of Multiple Species Resolved by Fluorescence Lifetime Recovery after Photobleaching (FLRAP)
  Ikumi Mori; Shun Terasaka; Shoichi Yamaguchi; Takuhiro Otosu
  Analytical Chemistry, 巻：96, 号：12, 開始ページ：4854, 終了ページ：4859, 2024年03月, ［査読有り］, ［最終著者, 責任著者］
  American Chemical Society (ACS), 研究論文（学術雑誌）
  DOI：https://doi.org/10.1021/acs.analchem.3c05181
  DOI ID：10.1021/acs.analchem.3c05181, ISSN：0003-2700, eISSN：1520-6882
• Theoretical and experimental OD-stretch vibrational spectroscopy of heavy water
  Tetsuyuki Takayama; Takuhiro Otosu; Shoichi Yamaguchi
  The Journal of Chemical Physics, 巻：160, 号：10, 2024年03月, ［査読有り］
  In view of the current situation in which the OD-stretch vibrational spectra have been scarcely computed with non-polarizable rigid D2O models, we investigate the IR and Raman spectra of D2O by using a newly-reported model TIP4P/2005-HW. From the comparison between the calculations and experimental data, we find the excellent performance of TIP4P/2005-HW for vibrational spectroscopy of D2O in the same manner as TIP4P/2005 for H2O, although one may still conveniently employ an alternative method that regards OH as putative OD to calculate the OD-stretch spectra with similar quality from TIP4P/2005 trajectories. We also demonstrate that the appropriate setting for the spectral simulation of D2O under the time-averaging approximation reflects the slower dynamics (i.e., slower motion of translation and rotation due to the heavier mass and stronger hydrogen bond) of D2O than H2O. Moreover, we show from the theoretical calculations that the established interpretation of the OH-stretch spectra of H2O is finely applicable to the OD-stretch of D2O.
  AIP Publishing, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1063/5.0200623
  DOI ID：10.1063/5.0200623, ISSN：0021-9606, eISSN：1089-7690
• Peripheral adsorption of polylysine on one leaflet of a lipid bilayer reduces the lipid diffusion of both leaflets
  Kosei Shimizu; Miyuki Sakaguchi; Shoichi Yamaguchi; Takuhiro Otosu
  Physical Chemistry Chemical Physics, 巻：26, 号：11, 開始ページ：8873, 終了ページ：8878, 2024年02月, ［査読有り］, ［最終著者, 責任著者］
  Leaflet-specific lipid diffusion analysis revealed that polylysine adsorption on one leaflet of a lipid bilayer is sensed by the opposite leaflet.
  Royal Society of Chemistry (RSC), 研究論文（学術雑誌）
  DOI：https://doi.org/10.1039/d3cp04882a
  DOI ID：10.1039/d3cp04882a, ISSN：1463-9076, eISSN：1463-9084
• Appraisal of TIP4P-type models at water surface
  Shoichi Yamaguchi; Tetsuyuki Takayama; Takuhiro Otosu
  The Journal of Chemical Physics, 巻：159, 号：17, 2023年11月, ［査読有り］
  In view of the current situation in which non-polarizable rigid water models have been scarcely examined against surface-specific properties, we appraise TIP4P-type models at the liquid water surface on the basis of heterodyne-detected sum frequency generation (HD-SFG) spectroscopy. We find in the HD-SFG spectrum of the water surface that the peak frequency of the hydrogen-bonded OH band, the half width at half maximum of the hydrogen-bonded OH band, and the full width at half maximum of the free OH band are best reproduced by TIP4P, TIP4P/Ew, and TIP4P/Ice, respectively, whereas it is already well known that TIP4P/2005 best reproduces the surface tension. These TIP4P-type models perform better at the water surface in terms of the present appraisal items than some polarizable models in the literature.
  AIP Publishing, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1063/5.0171999
  DOI ID：10.1063/5.0171999, ISSN：0021-9606, eISSN：1089-7690
• Transferability of vibrational spectroscopic map from TIP4P to TIP4P-like water models
  Tetsuyuki Takayama; Takuhiro Otosu; Shoichi Yamaguchi
  The Journal of Chemical Physics, 巻：158, 号：13, 2023年04月, ［査読有り］
  We computed the IR, Raman, and sum frequency generation spectra of water in the OH-stretch region by employing the quantum/classical mixed approach that consists of a vibrational spectroscopic map and molecular dynamics (MD) simulation. We carried out the MD simulation with the TIP4P, TIP4P/2005, and TIP4P/Ice models and applied the map designed for TIP4P by Skinner et al. to each MD trajectory. Although the map is not tuned for TIP4P-like models, TIP4P/2005 and TIP4P/Ice provide the best reproduction of the experimental vibrational spectra of liquid water and crystalline ice, respectively. This result demonstrates the transferability of the map from TIP4P to TIP4P/2005 and TIP4P/Ice, meaning that one can choose an appropriate TIP4P-like model to calculate the vibrational spectra of an aqueous system without rebuilding the map.
  AIP Publishing, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1063/5.0146084
  DOI ID：10.1063/5.0146084, ISSN：0021-9606, eISSN：1089-7690
• Experimental and Theoretical Heterodyne-Detected Sum Frequency Generation Spectroscopy of Isotopically Pure and Diluted Water Surfaces　　　　　　　　　　　　　　　
  Shoichi Yamaguchi; Tetsuyuki Takayama; Yuki Goto; Takuhiro Otosu; Takuma Yagasaki
  The Journal of Physical Chemistry Letters, 2022年10月, ［査読有り］
  研究論文（学術雑誌）
  DOI：https://doi.org/10.1021/acs.jpclett.2c02533
  DOI ID：10.1021/acs.jpclett.2c02533, ORCID：120487669
• Experimental and Theoretical Raman Spectroscopy of Isotopically Pure and Diluted Ice VI　　　　　　　　　　　　　　　
  Tetsuyuki Takayama; Kota Kishi; Takuhiro Otosu; Takuma Yagasaki; Shoichi Yamaguchi
  The Journal of Physical Chemistry C, 2022年10月, ［査読有り］
  研究論文（学術雑誌）
  DOI：https://doi.org/10.1021/acs.jpcc.2c05133
  DOI ID：10.1021/acs.jpcc.2c05133, ORCID：120088265
• Raman spectroscopy of isotopically pure and diluted high‐ and low‐density amorphous ices
  Senri Ishihara; Tetsuyuki Takayama; Miyuki Sakaguchi; Takuhiro Otosu; Takuma Yagasaki; Yoshiharu Suzuki; Shoichi Yamaguchi
  Journal of Raman Spectroscopy, 2022年02月, ［査読有り］
  Wiley, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1002/jrs.6322
  DOI ID：10.1002/jrs.6322, ISSN：0377-0486, eISSN：1097-4555
• Progress in phase-sensitive sum frequency generation spectroscopy
  Shoichi Yamaguchi; Takuhiro Otosu
  Physical Chemistry Chemical Physics, 巻：23, 号：34, 開始ページ：18253, 終了ページ：18267, 2021年06月, ［査読有り］
  Representative methods of surface-selective phase-sensitive sum frequency generation spectroscopy are reviewed in terms of interferometer implementation for optical heterodyne detection.
  Royal Society of Chemistry (RSC), 研究論文（学術雑誌）
  DOI：https://doi.org/10.1039/d1cp01994e
  DOI ID：10.1039/d1cp01994e, ISSN：1463-9076, eISSN：1463-9084
• Leaflet-specific Lipid Diffusion Revealed by Fluorescence Lifetime Correlation Analyses　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  CHEMISTRY LETTERS, 巻：49, 号：12, 開始ページ：1473, 終了ページ：1480, 2020年12月, ［査読有り］, ［招待有り］, ［筆頭著者］
  Cell membranes are fluidic, and the fluidic properties give unique biological functions to the membranes. Here, we review the application of fluorescence lifetime correlation analyses to elucidate the leaflet-specific lipid diffusion in a model cell membrane supported on a glass surface. Utilization of fluorescence lifetime enables us to discriminate the signals from each leaflet of the lipid bilayer and to analyze the lipid diffusion in a leaflet-specific manner. The results showed that the lipid diffusion in a proximal leaflet facing a glass surface is sensitive to the strength of the electrostatic interaction between the lipid headgroup and the glass surface whereas that in the distal one is insensitive to it. This clearly indicates that the interleaflet coupling is negligible. Because the strength of the interleaflet coupling is highly relevant to the cooperative formation of functional domains across the bilayer, this work provides an important insight into the lipid dynamics in cell membranes.
  CHEMICAL SOC JAPAN, 英語
  DOI：https://doi.org/10.1246/cl.200539
  DOI ID：10.1246/cl.200539, ISSN：0366-7022, eISSN：1348-0715, Web of Science ID：WOS:000598377200011
• Effect of electrostatic interaction on the leaflet-specific diffusion in a supported lipid bilayer revealed by fluorescence lifetime correlation analysis　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  PHYSICAL CHEMISTRY CHEMICAL PHYSICS, 巻：22, 号：3, 開始ページ：1242, 終了ページ：1249, 2020年01月, ［査読有り］, ［筆頭著者］
  A supported lipid bilayer (SLB) is now an indispensable tool to analyze the dynamical properties of biomembranes. However, the effect of a solid support on the leaflet-specific lipid dynamics in a SLB remains elusive, which hampers the further application of the SLB as a model biomembrane. Here, we performed the leaflet-specific lipid diffusion analysis by means of two-dimensional fluorescence lifetime correlation spectroscopy to elucidate the effect of the electrostatic interaction between lipid headgroups and a glass surface on the lipid diffusion in each leaflet of the SLB. The results clearly showed the correlation between the strength of the electrostatic interaction and the lipid diffusion in the proximal leaflet of the SLB facing a glass surface. In particular, the electrostatic attraction between the cationic lipids and a negatively charged glass surface enhanced the lipid diffusion in the proximal leaflet of the SLB, providing important implications for the lipid dynamics not only in the SLB but also in biomembranes.
  ROYAL SOC CHEMISTRY, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1039/c9cp05833h
  DOI ID：10.1039/c9cp05833h, ISSN：1463-9076, eISSN：1463-9084, Web of Science ID：WOS:000509371400030
• Multifocus Fluorescence Correlation Spectroscopy with Spatially Separated Excitation Beams　　　　　　　　　　　　　　　
  Takuhiro Otosu; Kunihiko Ishii; Tahei Tahara
  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, 巻：92, 号：9, 開始ページ：1495, 終了ページ：1502, 2019年09月, ［査読有り］, ［筆頭著者］
  We report on a new multifocus optical system developed for fluorescence correlation spectroscopy (FCS). This system utilizes a beamsplitter array to spatially separate excitation beams. Fluorescence from each spot is individually detected with a single-photon avalanche photodiode (SPAD) through a fiber bundle. The combined use of beamsplitters and multiple SPADs is beneficial to give a higher detection uniformity among focal spots than the existing multifocus FCS systems. Applications of this setup show that the variation of the optical parameters among seven focal spots are as small as similar to 5% and prove its high performance in FCS measurements. High efficiency of this system is also demonstrated in application to an advanced FCS method, two-dimensional fluorescence lifetime correlation spectroscopy. Along with the results of these applications, we describe a method for proper correction of the crosstalk effect in the multifocus setup.
  CHEMICAL SOC JAPAN, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1246/bcsj.20190109
  DOI ID：10.1246/bcsj.20190109, ISSN：0009-2673, eISSN：1348-0634, Web of Science ID：WOS:000482728000012
• Reduction of glass-surface charge density slows the lipid diffusion in the proximal leaflet of a supported lipid bilayer　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  JOURNAL OF CHEMICAL PHYSICS, 巻：151, 号：2, 2019年07月, ［査読有り］, ［筆頭著者］
  Understanding the effect of a solid support on the dynamical properties of a supported lipid bilayer (SLB) is a prerequisite for the applications of SLB as a model biomembrane. Here, we applied two-dimensional fluorescence lifetime correlation spectroscopy to examine the effect of solution pH on the diffusion of lipids in the proximal/distal leaflets of a zwitterionic SLB. Leaflet-specific diffusion analyses at various pH revealed that the diffusion of lipids in the proximal leaflet facing a glass surface becomes slower by decreasing pH with the transition pH of similar to 7.4. We attributed it to the reduction of the surface charge density of a glass support. Furthermore, the data clearly showed that the lipid diffusion in the distal leaflet facing a bulk solution is insensitive to the change in the diffusion property of the proximal leaflet. This reflects a weak interleaflet coupling between the proximal and distal leaflets of the SLB. Published under license by AIP Publishing.
  AMER INST PHYSICS, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1063/1.5103221
  DOI ID：10.1063/1.5103221, ISSN：0021-9606, eISSN：1089-7690, Web of Science ID：WOS:000486055700034
• Perspective on sum frequency generation spectroscopy of ice surfaces and interfaces　　　　　　　　　　　　　　　
  Shoichi Yamaguchi; Yudai Suzuki; Yuki Nojima; Takuhiro Otosu
  CHEMICAL PHYSICS, 巻：522, 開始ページ：199, 終了ページ：210, 2019年06月, ［査読有り］
  This perspective is focused on surfaces and interfaces of crystalline ice studied with sum frequency generation (SFG) spectroscopy by several research groups within these twenty years. The intrinsic interface selectivity of SFG has already enabled one to determine structural aspects of the ice surfaces and interfaces through vibrational signatures of the OH stretch. Here we describe why SFG is suitable for ice surface science and how SFG has revealed the structure and dynamics of dangling bonds, quasi-liquid layer, hydrogen-bond network, and ordered protons at the ice surfaces, ice/substrate interfaces, and ice films on metal surfaces. In particular, we compare SFG spectra of ice reported by different groups to examine consistency between their experiments. We elaborate current controversies on a few SFG studies of ice before presenting future outlook for ice surface science by SFG. Additionally in the appendix we briefly explain some technical issues that may help us resolve conflicts and make progress in the right way.
  ELSEVIER SCIENCE BV, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.chemphys.2019.03.005
  DOI ID：10.1016/j.chemphys.2019.03.005, ISSN：0301-0104, eISSN：1873-4421, Web of Science ID：WOS:000466713000027
• Quantifying the Diffusion of Lipids in the Proximal/Distal Leaflets of a Supported Lipid Bilayer by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  JOURNAL OF PHYSICAL CHEMISTRY B, 巻：122, 号：45, 開始ページ：10315, 終了ページ：10319, 2018年11月, ［査読有り］, ［筆頭著者］
  A supported lipid bilayer (SLB) is a versatile platform for examining the dynamical properties of biomembranes. However, the effect of a prerequisite solid substrate on the dynamics of a SLB remains very elusive. Especially, it is not clarified how the diffusivity of each leaflet in a SLB is affected by the SLB solid substrate interaction. In this study, we applied two-dimensional fluorescence lifetime correlation spectroscopy to a SLB for elucidating the diffusion of lipids in the proximal and distal leaflets of a SLB. We find that the autocorrelation curve of a fluorescent lipid in the proximal leaflet decays more slowly than that in the distal leaflet, meaning that the proximal leaflet is less diffusive. This result indicates stronger interaction between the proximal leaflet and a solid substrate.
  AMER CHEMICAL SOC, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1021/acs.jpcb.8b08614
  DOI ID：10.1021/acs.jpcb.8b08614, ISSN：1520-6106, Web of Science ID：WOS:000451101100006
• Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  MOLECULES, 巻：23, 号：11, 2018年11月, ［査読有り］, ［筆頭著者］
  We review the basic concepts and recent applications of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which is the extension of fluorescence correlation spectroscopy (FCS) to analyze the correlation of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime is sensitive to the microenvironment and can be a "molecular ruler" when combined with FRET. Utilization of fluorescence lifetime in 2D FLCS thus enables us to quantify the inhomogeneity of the system and the interconversion dynamics among different species with a higher time resolution than other single-molecule techniques. Recent applications of 2D FLCS to various biological systems demonstrate that 2D FLCS is a unique and promising tool to quantitatively analyze the microsecond conformational dynamics of macromolecules at the single-molecule level.
  MDPI, 英語
  DOI：https://doi.org/10.3390/molecules23112972
  DOI ID：10.3390/molecules23112972, eISSN：1420-3049, Web of Science ID：WOS:000451641900244
• Total Internal Reflection Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  JOURNAL OF PHYSICAL CHEMISTRY B, 巻：122, 号：22, 開始ページ：5758, 終了ページ：5764, 2018年06月, ［査読有り］
  Fluorescence lifetime correlation analysis is becoming a powerful tool to understand the conformational heterogeneity of biomolecules and their dynamics with an unprecedented detection sensitivity and time resolution. However, its application to the study of biomembranes is very limited. Here, we report on two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) in combination with total internal reflection (TIR) microscopy (TIR 2D-FLCS). High depth resolution in TIR microscopy and species specific correlation analysis in 2D FLCS give us the opportunity to selectively analyze molecules in or on a supported lipid bilayer, a model biomembrane formed on the glass surface. Feasibility experiments performed in this study clearly demonstrated that TIR 2D-FLCS has a potential to selectively analyze the diffusion and the conformational dynamics of proteins peripherally bound on the membrane in the presence of substantial amounts of unbound molecules in the bulk phase.
  AMER CHEMICAL SOC, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1021/acs.jpcb.8b01176
  DOI ID：10.1021/acs.jpcb.8b01176, ISSN：1520-6106, SCOPUS ID：85048247541, Web of Science ID：WOS:000435019600004
• Local pH at the surface of hen egg white lysozyme　　　　　　　　　　　　　　　
  Takuhiro Otosu; Kaito Kobayashi; Shoichi Yamaguchi
  CHEMICAL PHYSICS LETTERS, 巻：693, 開始ページ：165, 終了ページ：169, 2018年02月, ［査読有り］
  The microenvironment at the surface of hen-egg-white lysozyme (HEWL) was examined by analyzing the change in pK(a) of fluorescein isothiocyanate (FITC) upon binding to the N-terminus of HEWL. The result showed that the local pH at the HEWL surface is higher than the bulk pH. Furthermore, the data showed that the difference between the local and bulk pH becomes larger with decreasing pH, suggesting HEWL repels more protons at lower pH. Because the local pH affects the protonation states of functional amino-acids at the protein surface, the results provide the fundamental insight into the microenvironment at the protein surface. (C) 2018 Elsevier B.V. All rights reserved.
  ELSEVIER SCIENCE BV, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.cplett.2018.01.026
  DOI ID：10.1016/j.cplett.2018.01.026, ISSN：0009-2614, eISSN：1873-4448, SCOPUS ID：85040701289, Web of Science ID：WOS:000424631900026
• Communication: Development of standing evanescent-wave fluorescence correlation spectroscopy and its application to the lateral diffusion of lipids in a supported lipid bilayer　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  JOURNAL OF CHEMICAL PHYSICS, 巻：147, 号：4, 開始ページ：041101, 2017年07月, ［査読有り］, ［筆頭著者］
  We present standing evanescent-wave fluorescence correlation spectroscopy (SEW-FCS). This technique utilizes the interference of two evanescent waves which generates a standing evanescent-wave. Fringe-pattern illumination created by a standing evanescent-wave enables us to measure the diffusion coefficients of molecules with a super-resolution corresponding to one fringe width. Because the fringe width can be reliably estimated by a simple procedure, utilization of fringes is beneficial to quantitatively analyze the slow diffusion of molecules in a supported lipid bilayer (SLB), a model biomembrane formed on a solid substrate, with the timescale relevant for reliable FCS analysis. Furthermore, comparison of the data between SEW-FCS and conventional total-internal reflection FCS, which can also be performed by the SEW-FCS instrument, effectively eliminates the artifact due to afterpulsing of the photodiode detector. The versatility of SEW-FCS is demonstrated by its application to various SLBs. Published by AIP Publishing.
  AMER INST PHYSICS, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1063/1.4985871
  DOI ID：10.1063/1.4985871, ISSN：0021-9606, eISSN：1089-7690, Web of Science ID：WOS:000406347200002
• Highly Heterogeneous Nature of the Native and Unfolded States of the B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy　　　　　　　　　　　　　　　
  Takuhiro Otosu; Kunihiko Ishii; Hiroyuki Oikawa; Munehito Arai; Satoshi Takahashi; Tahei Tahara
  JOURNAL OF PHYSICAL CHEMISTRY B, 巻：121, 号：22, 開始ページ：5463, 終了ページ：5473, 2017年06月, ［査読有り］
  Elucidating the protein folding mechanism is crucial to understand how proteins acquire their unique structures to realize various biological functions. With this aim, the folding/unfolding of small globular proteins has been extensively studied. Interestingly, recent studies have revealed that even such small prote(i)ns represent considerably complex processes. In this study, we examined the folding/unfolding process of a small a-helical protein, the B domain of protein A (BdpA), at equilibrium using two-dimensional fluorescence lifetime correlation spectroscopy with 10 mu s time resolution. The results showed that although the BdpA is a two-state folder, both the native and unfolded states are highly heterogeneous and the conformational conversion within each ensemble occurs within 10 mu s. Furthermore, it was shown that the average structures of both ensembles gradually change and become more elongated as the denaturant concentration increases. The analysis on two mutants suggested that fraying of the N-terminal helix is the origin of the inhomogeneity of the native state. Because the direct observation of the ensemble nature of the native state at the single-molecule level has not been reported, the data obtained in this study give new insights into complex conformational properties of small proteins.
  AMER CHEMICAL SOC, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1021/acs.jpcb.7b00546
  DOI ID：10.1021/acs.jpcb.7b00546, ISSN：1520-6106, Web of Science ID：WOS:000403191700001
• Standing evanescent-wave fluorescence correlation spectroscopy for analyzing the translational diffusion in bio-membranes　　　　　　　　　　　　　　　
  Takuhiro Otosu; Shoichi Yamaguchi
  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 巻：252, 2016年08月, ［査読有り］, ［筆頭著者］
  AMER CHEMICAL SOC, 英語
  ISSN：0065-7727, Web of Science ID：WOS:000431460201108
• Microsecond protein dynamics observed at the single-molecule level　　　　　　　　　　　　　　　
  Takuhiro Otosu; Kunihiko Ishii; Tahei Tahara
  NATURE COMMUNICATIONS, 巻：6, 2015年07月, ［査読有り］, ［筆頭著者］
  How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.
  NATURE PUBLISHING GROUP, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1038/ncomms8685
  DOI ID：10.1038/ncomms8685, ISSN：2041-1723, Web of Science ID：WOS:000358858100029
• Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy on the Folding Mechanism of B Domain of Protein A　　　　　　　　　　　　　　　
  Takuhiro Otosu; Kunihiko Ishii; Hiroyuki Oikawa; Munehito Arai; Satoshi Takahashi; Tahei Tahara
  BIOPHYSICAL JOURNAL, 巻：108, 号：2, 開始ページ：501A, 終了ページ：501A, 2015年01月, ［査読有り］
  CELL PRESS, 英語
  DOI：https://doi.org/10.1016/j.bpj.2014.11.2747
  DOI ID：10.1016/j.bpj.2014.11.2747, ISSN：0006-3495, eISSN：1542-0086, Web of Science ID：WOS:000362849600141
• 1P055 二次元蛍光寿命相関分光法によるBdpA折りたたみ機構解明に向けた研究 : 2つのBdpA変異体による包括的解析(01C. 蛋白質 : 物性,ポスター,第52回日本生物物理学会年会(2014年度))　　　　　　　　　　　　　　　
  Otosu Takuhiro; Ishii Kunihiko; Oikawa Hiroyuki; Arai Munehito; Takahashi Satoshi; Tahara Tahei
  生物物理, 巻：54, 号：1, 開始ページ：S150, 2014年
  一般社団法人 日本生物物理学会, 英語
  DOI：https://doi.org/10.2142/biophys.54.S150_1
  DOI ID：10.2142/biophys.54.S150_1, CiNii Articles ID：110009932279
• Microsecond Conformational Dynamics of Cytochrome C Revealed by Two-Dimentional Fluorescence Lifetime Correlation Spectroscopy　　　　　　　　　　　　　　　
  Takuhiro Otosu; Kunihiko Ishii; Tahei Tahara
  BIOPHYSICAL JOURNAL, 巻：106, 号：2, 開始ページ：459A, 終了ページ：459A, 2014年01月, ［査読有り］
  CELL PRESS, 英語
  DOI：https://doi.org/10.1016/j.bpj.2013.11.2603
  DOI ID：10.1016/j.bpj.2013.11.2603, ISSN：0006-3495, eISSN：1542-0086, Web of Science ID：WOS:000337000402557
• Note: Simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis　　　　　　　　　　　　　　　
  Takuhiro Otosu; Kunihiko Ishii; Tahei Tahara
  REVIEW OF SCIENTIFIC INSTRUMENTS, 巻：84, 号：3, 2013年03月, ［査読有り］
  The counting-rate dependence of the temporal response of single photon avalanche diodes (SPADs) is a critical issue for the accurate determination of the fluorescence lifetime. In this study, the response of SPADs was examined with analyzing the time interval of the detected photons. The results clearly show that the shift of the detection timing causes the counting-rate dependence of the temporal response, and this timing shift is solely determined by the time interval from the preceding photon. We demonstrate that this timing instability is readily calibrated by utilizing the macrotime data taken with the time-tag mode that is implemented in the time-correlated single photon counting modules. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4794769]
  AMER INST PHYSICS, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1063/1.4794769
  DOI ID：10.1063/1.4794769, ISSN：0034-6748, eISSN：1089-7623, Web of Science ID：WOS:000316966200057
• 3P052 二次元蛍光寿命相関分光法によるBdpA変性状態における構造ダイナミクスの解析(01C.蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))　　　　　　　　　　　　　　　
  Otosu Takuhiro; Ishii Kunihiko; Oikawa Hiroyuki; Arai Munehito; Takahashi Satoshi; Tahara Tahei
  生物物理, 巻：53, 号：1, 開始ページ：S220, 2013年
  一般社団法人 日本生物物理学会, 英語
  DOI：https://doi.org/10.2142/biophys.53.S220_4
  DOI ID：10.2142/biophys.53.S220_4, CiNii Articles ID：110009819856
• Multiple conformational state of human serum albumin around single tryptophan residue at various pH revealed by time-resolved fluorescence spectroscopy　　　　　　　　　　　　　　　
  Takuhiro Otosu; Etsuko Nishimoto; Shoji Yamashita
  JOURNAL OF BIOCHEMISTRY, 巻：147, 号：2, 開始ページ：191, 終了ページ：200, 2010年02月, ［査読有り］, ［筆頭著者］
  Human serum albumin (HSA) plays important roles in transport of fatty acids and binding a variety of drugs and organic compounds in the circulatory system. This protein experiences several conformational transitions by the change of pH, and the resulting conformations were essential for completing the physiological roles in vivo. Steady-state and time-resolved fluorescence spectroscopy was applied to single tryptophan residue solely arranged in HSA to study subtle conformational change around single tryptophan residue in HSA at various pH. The results showed the characteristic feature of local conformation around tryptophan residue in domain II responding to the change in entire structure. The study of time-resolved area-normalized fluorescence emission spectra (TRANES) also showed the peculiar dielectric property of water molecule trapped nearby tryptophan residue depending on pH. These results suggested that microenvironment around tryptophan residue was tightly packed at acidic and basic pH although entire conformation was loosened.
  OXFORD UNIV PRESS, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1093/jb/mvp175
  DOI ID：10.1093/jb/mvp175, ISSN：0021-924X, Web of Science ID：WOS:000274344400006
• Fluorescence Decay Characteristics of Indole Compounds Revealed by Time-Resolved Area-Normalized Emission Spectroscopy　　　　　　　　　　　　　　　
  Takuhiro Otosu; Etsuko Nishimoto; Shoji Yamashita
  JOURNAL OF PHYSICAL CHEMISTRY A, 巻：113, 号：12, 開始ページ：2847, 終了ページ：2853, 2009年03月, ［査読有り］
  Time-resolved fluorescence spectroscopy of tryptophan residue has been extensively applied to the studies on structure-function relationships of protein. Regardless of this, the fluorescence decay mechanism and kinetics of tryptophan residue in many proteins still remains unclear. Previous studies have demonstrated that conformational heterogeneity and relaxation dynamics are both involved in the peculiar multiexponential decay kinetics in subnanosecond resolution. In the present study, we characterized the fluorescence decay property of six indole compounds in glycerol by resolving the contribution of conformational heterogeneity and relaxation dynamics. We applied the time-resolved area-normalized fluorescence emission spectrum (TRANES) method for the fluorescence decay analysis. The results of TRANES, time-dependent shift of fluorescence spectral center of gravity, and fluorescence decay simulation demonstrated that the dielectric relaxation process independent of intrinsic rotamer/conformer and the individual fluorescence lifetime gives the peculiarity to the fluorescence decay of indole compounds. These results confirmed that TRANES and time-dependent spectral shift analysis are potent methods to resolve the origin of multiexponential decay kinetics of tryptophyl fluorescence in protein.
  AMER CHEMICAL SOC, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1021/jp8078937
  DOI ID：10.1021/jp8078937, ISSN：1089-5639, Web of Science ID：WOS:000264348800024
• The unfolding of alpha-momorcharin proceeds through the compact folded intermediate　　　　　　　　　　　　　　　
  Yukihiro Fukunaga; Etsuko Nishimoto; Takuhiro Otosu; Yasutaka Murakami; Shoji Yamashita
  JOURNAL OF BIOCHEMISTRY, 巻：144, 号：4, 開始ページ：457, 終了ページ：466, 2008年10月, ［査読有り］
  The unfolding of alpha-momorcharin was systematically investigated using steady-state and time-resolved tryptophan fluorescence, circular dichroism and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. These spectroscopic studies demonstrated that alpha-momorcharin unfolded through a compact folded intermediate state. The content of alpha-helix was increased, Trp192 approached closer to the side of active site and its rotational motion was restricted by being equilibrated with 23 M of guanidine hydrochloride. Furthermore, the binding of ANS with alpha-momorcharin was more suppressed to show that the hydrophobic parts would not be accessed to the protein surface but rather be sealed off in this specific conformation state. These results suggest that the structure of alpha-momorcharin holds the more compact conformation as an incipient state for unfolding, which is the sharp contrast to beta-momorcharin that gives the characteristics of the generally known molten globule state.
  OXFORD UNIV PRESS, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1093/jb/mvn088
  DOI ID：10.1093/jb/mvn088, ISSN：0021-924X, Web of Science ID：WOS:000259770200006
• Heterogeneous packing in the folding/unfolding intermediate state of bitter gourd trypsin inhibitor　　　　　　　　　　　　　　　
  Daisuke Takahashi; Shuzo Matsumoto; Etsuko Nishimoto; Takuhiro Otosu; Shoji Yamashita
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 巻：72, 号：6, 開始ページ：1498, 終了ページ：1505, 2008年06月, ［査読有り］
  The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2-3m guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.
  TAYLOR & FRANCIS LTD, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1271/bbb.80009
  DOI ID：10.1271/bbb.80009, ISSN：0916-8451, eISSN：1347-6947, Web of Science ID：WOS:000257305300012
• 2P-091 蛍光法による酸性条件下での蛋白質変性中間体解析(蛋白質・物性(2),第46回日本生物物理学会年会)　　　　　　　　　　　　　　　
  Otosu Takuhiro; Nishimoto Etsuko; Murakami Yasutaka; Yamashita Shoji
  生物物理, 巻：48, 開始ページ：S89, 2008年
  一般社団法人 日本生物物理学会, 英語
  DOI：https://doi.org/10.2142/biophys.48.S89_2
  DOI ID：10.2142/biophys.48.S89_2, CiNii Articles ID：110007013592
• 2P-087 リフォールディングモモルチャリンの構造とダイナミクス(蛋白質・物性(2),第46回日本生物物理学会年会)　　　　　　　　　　　　　　　
  Murakami Yasutaka; Nishimoto Etsuko; Otosu Takuhiro; Yamashita Shoji
  生物物理, 巻：48, 開始ページ：S88, 2008年
  一般社団法人 日本生物物理学会, 英語
  DOI：https://doi.org/10.2142/biophys.48.S88_5
  DOI ID：10.2142/biophys.48.S88_5, CiNii Articles ID：110007013588
• Spectrally and time-resolved fluorescence spectroscopic study on melittin-calmodulin interaction　　　　　　　　　　　　　　　
  Takuhiro Otosu; Etsuko Nishimoto; Shoji Yamashita
  JOURNAL OF BIOCHEMISTRY, 巻：142, 号：5, 開始ページ：655, 終了ページ：661, 2007年11月, ［査読有り］
  The origin of multi-exponential fluorescence decay property of tryptophan (Trp) in protein has been in controversy, and dielectric relaxation is thought to be one of the most plausible candidates of that origin. In this study, we studied melittin-calmodulin interaction on the concept of dielectric relaxation by spectrally and time-resolved fluorescence spectroscopy. Trp residue in melittin demonstrated drastic change in its dielectric relaxation rate and scale by binding with calmodulin. Expected change of relaxation rate suggested that dielectric relaxation accounts for multi-exponential property of fluorescence decay. We also examined the time variation of radiative and non-radiative decay rates. That result demonstrated the distinct difference profiles of non-radiative decay rate of Trp in melittin and the complex.
  OXFORD UNIV PRESS, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1093/jb/mvm180
  DOI ID：10.1093/jb/mvm180, ISSN：0021-924X, Web of Science ID：WOS:000253630200014
• 2P046 時間分割蛍光法によるRNase T1中Tryptophan残基の複数の誘電緩和特性に関する研究(蛋白質(構造・構造機能相関),口頭発表,第45回日本生物物理学会年会)　　　　　　　　　　　　　　　
  乙須 拓洋; 西本 悦子; 宮崎 義史; 山下 昭二
  生物物理, 巻：47, 開始ページ：S124, 2007年
  一般社団法人 日本生物物理学会, 英語
  DOI：https://doi.org/10.2142/biophys.47.S124_3
  DOI ID：10.2142/biophys.47.S124_3, CiNii Articles ID：110006562022
• The partially unfolded state of beta-momorcharin characterized with steady-state and time-resolved fluorescence studies　　　　　　　　　　　　　　　
  Yukihiro Fukunaga; Etsuko Nishimoto; Katsumi Yamashita; Takuhiro Otosu; Shoji Yamashita
  JOURNAL OF BIOCHEMISTRY, 巻：141, 号：1, 開始ページ：9, 終了ページ：18, 2007年01月, ［査読有り］
  The specific conformation of partially unfolded state of P-momorcharin was characterized through the steady-state and time-resolved fluorescence spectroscopic studies on a single Trp-190 which located adjacently to the active site. The content of secondary structure was retained, the binding of ANS was remarkably enhanced, and the correlation time of entire protein rotation was prolonged at the partially unfolded state formed by being equilibrated with the mild concentration of guanidine hydrochloride. The time-resolved fluorescence depolarization and excitation energy transfer analysis suggest that Trp-190 approached 2 angstrom closer to Tyr-70 and was hidden from the exposure to the protein surface, while the rotational correlation time and freedom of its segmental motion were shortened and enhanced, respectively. These results suggest that the transient folding/unfolding intermediate state of P-momorcharin adopt the specific conformation at the vicinity of the active site, although it exhibits very similar properties with those of the generally known molten-globule state.
  JAPANESE BIOCHEMICAL SOC, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1093/jb/mvm002
  DOI ID：10.1093/jb/mvm002, ISSN：0021-924X, Web of Science ID：WOS:000244866600002
• 2P010 Fluorescence spectroscopic study on the CaM-target protein interaction-Interaction with melittin(29. Protein structure and dynamics (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)　　　　　　　　　　　　　　　
  Otosu Takuhiro; Nishimoto Etsuko; Fukunaga Yukihiro; Miyazaki Yoshifumi; Yamashita Shoji
  生物物理, 巻：46, 号：2, 開始ページ：S298, 2006年
  一般社団法人 日本生物物理学会, 英語
  DOI：https://doi.org/10.2142/biophys.46.S298_2
  DOI ID：10.2142/biophys.46.S298_2, CiNii Articles ID：110006193971
• 1P051 カルモデュリン-グルタミン酸デカルボキシラーゼに見る蛋白質間相互作用(蛋白質 B) 構造・機能相関))　　　　　　　　　　　　　　　
  乙須 拓洋; 松元 俊彦; 西本 悦子; 山下 昭二; 山浦 泉
  生物物理, 巻：45, 開始ページ：S44, 2005年
  一般社団法人 日本生物物理学会, 日本語
  DOI：https://doi.org/10.2142/biophys.45.S44_3
  DOI ID：10.2142/biophys.45.S44_3, CiNii Articles ID：110004571032

• Advanced Photon Counting: Applications, Methods, Instrumentation　　　　　　　　　　　　　　　
  Ishii Kunihiko; Otosu Takuhiro; Tahara Tahei, ［分担執筆］, Chapter 9 Lifetime-weighted FCS and 2D FLCS: Advanced application of time-tagged TCSPC
  Springer, 2015年
  英語
  ISBN：9783319156361

• 新規分光計測による混合脂質二重膜の新たな相境界の探索　　　　　　　　　　　　　　　
  日本学術振興会, 科学研究費助成事業, 基盤研究(C), 2023年04月 - 2026年03月
  乙須 拓洋, 埼玉大学
  配分額（総額）：4550000, 配分額（直接経費）：3500000, 配分額（間接経費）：1050000
  課題番号：23K04662
• アミロイド形成を促す脂質‐蛋白質間相互作用の分子機構解明　　　　　　　　　　　　　　　
  日本学術振興会, 科学研究費助成事業, 基盤研究(B), 2019年04月01日 - 2023年03月31日
  乙須 拓洋; 山口 祥一, 埼玉大学
  配分額（総額）：15600000, 配分額（直接経費）：12000000, 配分額（間接経費）：3600000
  本研究ではモデル生体膜を用い，脂質二重膜上で促進されるアミロイド形成の分子機構理解を目的としている．2021年度はアミロイド形成蛋白質として知られているIAPPを用い，アミロイド感受性色素であるチオフラビンTを用いた凝集過程の追跡，ならびに蛍光ラベルしたIAPPを用いたIAPP拡散速度変化の追跡を行った．前者の実験の結果，IAPPはアミロイド形成開始後約70分間はアミロイドの形成は起こらず，その後加速度的にアミロイド形成が促進することが明らかとなった．
  この点についてより詳細に調べるために，蛍光ラベルしたIAPPを用いて，アミロイド形成開始後異なる経過時間での蛍光相関分光法によるIAPP拡散速度計測を行った．IAPPの拡散速度はIAPPの凝集サイズに敏感であることから，この測定により開始後70分間の間にどのような構造変化，またオリゴマーへの転移が起こっているかを調べることが可能となる．計測の結果，IAPPの拡散速度は70分の間ほとんど変化が見られなかった．この結果は，IAPPがアミロイド形成に至る前に形成するオリゴマーの分子サイズ（流体力学的半径）はモノマーとほとんど区別ができないことを示唆するものとなった．IAPPが構造変化を伴わずにオリゴマーとなった場合流体力学的半径の変化が起こることから，今回の結果はIAPPオリゴマーにおいてIAPPはモノマー時よりもよりコンパクトは構造へと転移している事を示唆する結果となった．
  課題番号：19H02669
• 膜融合の分子機構解明に向けた脂質二重膜間相互作用の分光学的研究　　　　　　　　　　　　　　　
  日揮実吉奨学会, 日揮実吉奨学会研究助成金, 2020年09月 - 2022年08月
  埼玉大学, 研究代表者
• 新規光技術に基づく界面選択的流速計測法の開発と応用　　　　　　　　　　　　　　　
  日本学術振興会, 科学研究費助成事業, 挑戦的研究(萌芽), 2017年06月30日 - 2019年03月31日
  乙須 拓洋, 埼玉大学
  配分額（総額）：6110000, 配分額（直接経費）：4700000, 配分額（間接経費）：1410000
  固液界面に代表される様々な界面近傍での物質の流れがバルクの流れとどう異なるかを理解することは，界面での効率的な化学反応の制御，血管内壁近傍での血液の流れの理解，などあらゆる分野における研究課題について重要な知見を与える．本研究では申請者が細胞膜研究を目的として開発を行った，エバネッセント光の干渉を利用する新規分光装置を界面での流速計測に応用し，高い精度での界面選択的流速計測法の確立を目指した．
  課題番号：17K19097
• 蛍光寿命の相関解析による蛋白質‐リガンド複合体形成機構解明　　　　　　　　　　　　　　　
  日本学術振興会, 科学研究費助成事業, 若手研究(B), 2015年04月01日 - 2017年03月31日
  乙須 拓洋, 埼玉大学
  配分額（総額）：4160000, 配分額（直接経費）：3200000, 配分額（間接経費）：960000
  蛋白質の構造揺らぎは生体機能発現と深くかかわっていることから，近年盛んに研究が行われている．本研究では平衡状態での構造揺らぎ解析の重要なツールである二次元蛍光寿命相関分光法を非平衡状態ダイナミクス研究に応用可能な手法へと拡張することを目的とした研究を行った．特に本研究ではリガンドの結合により誘起される蛋白質の構造変化追跡を目的とし，ケージド化合物の光解離に基づくリガンド放出を可能とする紫外光パルスと，蛋白質の構造変化追跡用の可視光パルスを同軸入射する新たな蛍光寿命相関分光装置の立ち上げに成功した．
  課題番号：15K17814