Performance information

• 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, Volume：27, Number：9, First page：4944, Last page：4949, Feb. 2025, [Reviewed], [Lead, Corresponding]
  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), Scientific journal
  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, Volume：161, Number：23, Dec. 2024, [Reviewed]
  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, Scientific journal
  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, Volume：96, Number：12, First page：4854, Last page：4859, Mar. 2024, [Reviewed], [Last, Corresponding]
  American Chemical Society (ACS), Scientific journal
  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, Volume：160, Number：10, Mar. 2024, [Reviewed]
  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, Scientific journal
  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, Volume：26, Number：11, First page：8873, Last page：8878, Feb. 2024, [Reviewed], [Last, Corresponding]
  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), Scientific journal
  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, Volume：159, Number：17, Nov. 2023, [Reviewed]
  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, Scientific journal
  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, Volume：158, Number：13, Apr. 2023, [Reviewed]
  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, Scientific journal
  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, Oct. 2022, [Reviewed]
  Scientific journal
  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, Oct. 2022, [Reviewed]
  Scientific journal
  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, Feb. 2022, [Reviewed]
  Wiley, Scientific journal
  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, Volume：23, Number：34, First page：18253, Last page：18267, Jun. 2021, [Reviewed]
  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), Scientific journal
  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, Volume：49, Number：12, First page：1473, Last page：1480, Dec. 2020, [Reviewed], [Invited], [Lead]
  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, English
  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, Volume：22, Number：3, First page：1242, Last page：1249, Jan. 2020, [Reviewed], [Lead]
  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, English, Scientific journal
  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, Volume：92, Number：9, First page：1495, Last page：1502, Sep. 2019, [Reviewed], [Lead]
  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, English, Scientific journal
  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, Volume：151, Number：2, Jul. 2019, [Reviewed], [Lead]
  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, English, Scientific journal
  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, Volume：522, First page：199, Last page：210, Jun. 2019, [Reviewed]
  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, English, Scientific journal
  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, Volume：122, Number：45, First page：10315, Last page：10319, Nov. 2018, [Reviewed], [Lead]
  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, English, Scientific journal
  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, Volume：23, Number：11, Nov. 2018, [Reviewed], [Lead]
  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, English
  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, Volume：122, Number：22, First page：5758, Last page：5764, Jun. 2018, [Reviewed]
  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, English, Scientific journal
  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, Volume：693, First page：165, Last page：169, Feb. 2018, [Reviewed]
  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, English, Scientific journal
  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, Volume：147, Number：4, First page：041101, Jul. 2017, [Reviewed], [Lead]
  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, English, Scientific journal
  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, Volume：121, Number：22, First page：5463, Last page：5473, Jun. 2017, [Reviewed]
  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, English, Scientific journal
  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, Volume：252, Aug. 2016, [Reviewed], [Lead]
  AMER CHEMICAL SOC, English
  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, Volume：6, Jul. 2015, [Reviewed], [Lead]
  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, English, Scientific journal
  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, Volume：108, Number：2, First page：501A, Last page：501A, Jan. 2015, [Reviewed]
  CELL PRESS, English
  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 Study of BdpA folding by two-dimensional fluorescence lifetime correlation spectroscopy : Comprehensive analysis of two BdpA mutants(01C. Protein : Property,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))　　　　　　　　　　　　　　　
  Otosu Takuhiro; Ishii Kunihiko; Oikawa Hiroyuki; Arai Munehito; Takahashi Satoshi; Tahara Tahei
  Seibutsu Butsuri, Volume：54, Number：1, First page：S150, 2014
  The Biophysical Society of Japan General Incorporated Association, English
  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, Volume：106, Number：2, First page：459A, Last page：459A, Jan. 2014, [Reviewed]
  CELL PRESS, English
  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, Volume：84, Number：3, Mar. 2013, [Reviewed]
  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, English, Scientific journal
  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 Two-dimensional fluorescence lifetime correlation spectroscopy on the conformational dynamics of the unfolded state of BdpA(01A. Protein: Structure,Poster)　　　　　　　　　　　　　　　
  Otosu Takuhiro; Ishii Kunihiko; Oikawa Hiroyuki; Arai Munehito; Takahashi Satoshi; Tahara Tahei
  Seibutsu Butsuri, Volume：53, Number：1, First page：S220, 2013
  The Biophysical Society of Japan General Incorporated Association, English
  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, Volume：147, Number：2, First page：191, Last page：200, Feb. 2010, [Reviewed], [Lead]
  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, English, Scientific journal
  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, Volume：113, Number：12, First page：2847, Last page：2853, Mar. 2009, [Reviewed]
  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, English, Scientific journal
  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, Volume：144, Number：4, First page：457, Last page：466, Oct. 2008, [Reviewed]
  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, English, Scientific journal
  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, Volume：72, Number：6, First page：1498, Last page：1505, Jun. 2008, [Reviewed]
  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, English, Scientific journal
  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 The fluorescence spectroscopic characterization of the protein folding/unfolding intermediate state in the acidic condition(The 46th Annual Meeting of the Biophysical Society of Japan)　　　　　　　　　　　　　　　
  Otosu Takuhiro; Nishimoto Etsuko; Murakami Yasutaka; Yamashita Shoji
  Seibutsu Butsuri, Volume：48, First page：S89, 2008
  The Biophysical Society of Japan General Incorporated Association, English
  DOI：https://doi.org/10.2142/biophys.48.S89_2
  DOI ID：10.2142/biophys.48.S89_2, CiNii Articles ID：110007013592
• 2P-087 The conformation and dynamics of the refolded momorcharins(The 46th Annual Meeting of the Biophysical Society of Japan)　　　　　　　　　　　　　　　
  Murakami Yasutaka; Nishimoto Etsuko; Otosu Takuhiro; Yamashita Shoji
  Seibutsu Butsuri, Volume：48, First page：S88, 2008
  The Biophysical Society of Japan General Incorporated Association, English
  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, Volume：142, Number：5, First page：655, Last page：661, Nov. 2007, [Reviewed]
  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, English, Scientific journal
  DOI：https://doi.org/10.1093/jb/mvm180
  DOI ID：10.1093/jb/mvm180, ISSN：0021-924X, Web of Science ID：WOS:000253630200014
• 2P046 Time-resolved fluorescence study on multiple dielectric relaxation properties of single tryptophan residue in RNase T1(Proteins-structure and structure-function relationship,Oral Presentations)　　　　　　　　　　　　　　　
  Otosu Takuhiro; Nishimoto Etsuko; Miyazaki Yoshifumi; Yamashita Shoji
  Seibutsu Butsuri, Volume：47, First page：S124, 2007
  The Biophysical Society of Japan General Incorporated Association, English
  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, Volume：141, Number：1, First page：9, Last page：18, Jan. 2007, [Reviewed]
  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, English, Scientific journal
  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
  Seibutsu Butsuri, Volume：46, Number：2, First page：S298, 2006
  The Biophysical Society of Japan General Incorporated Association, English
  DOI：https://doi.org/10.2142/biophys.46.S298_2
  DOI ID：10.2142/biophys.46.S298_2, CiNii Articles ID：110006193971
• 1P051 Protein-protein interaction between calmodulin and glutamic acid decarboxylase　　　　　　　　　　　　　　　
  Otosu T.; Matsumoto T.; Nishimoto E.; Yamashita S.; Yamaura I.
  Seibutsu Butsuri, Volume：45, First page：S44, 2005
  The Biophysical Society of Japan General Incorporated Association, Japanese
  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, [Contributor], Chapter 9 Lifetime-weighted FCS and 2D FLCS: Advanced application of time-tagged TCSPC
  Springer, 2015
  English
  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　　　　　　　　　　　　　　　
  Apr. 2023 - Mar. 2026
  Grant amount(Total)：4550000, Direct funding：3500000, Indirect funding：1050000
  Grant number：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　　　　　　　　　　　　　　　
  01 Apr. 2019 - 31 Mar. 2023
  Grant amount(Total)：15600000, Direct funding：12000000, Indirect funding：3600000
  Grant number：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　　　　　　　　　　　　　　　
  Sep. 2020 - Aug. 2022
  Principal investigator
• Development and application of novel flow velocimetry at the solid-liquid interface　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Research (Exploratory), 30 Jun. 2017 - 31 Mar. 2019
  Otosu Takuhiro, Saitama University
  Grant amount(Total)：6110000, Direct funding：4700000, Indirect funding：1410000
  Understanding the flow of particles at the solid-liquid interface has important implications on the regulation of the effective chemical reaction at the interface as well as the elucidation of blood flow at the inner surface of blood vessel. In the present study, I aimed at developing new optical technique for flow velocity measurement at the solid-liquid interface.
  For that purpose, I applied a novel fluorescence microscope system that I recently developed for biomembrane study. This system utilizes the interference of evanescent waves to excite samples. The fringe pattern (standing evanescent-wave) generated by the interference of the evanescent waves enables us to analyze the flow velocity with high spatial resolution beyond the diffraction limit. To apply this new technique to the flow-velocity measurement, I constructed surface-sensitive flow velocimetry by combining standing evanescent-wave fluorescence microscope with microfluidic device.
  Grant number：17K19097
• Protein-ligand complex formation revealed by fluorescence lifetime-correlation analysis　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists (B), 01 Apr. 2015 - 31 Mar. 2017
  OTOSU Takuhiro, Saitama University
  Grant amount(Total)：4160000, Direct funding：3200000, Indirect funding：960000
  Conformational fluctuations of proteins are highly relevant to their biological functions. To this aim, we developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) which analyze the conformational dynamics of biomolecules in an equilibrium by the correlation analysis of fluorescence lifetime. The aim of this study is to extend 2D FLCS to analyze the conformational dynamics in a non-equilibrium condition. For this purpose, I developed new 2D FLCS instrument which utilizes two laser pulses (UV pulse and visible pulse). UV pulse is used for uncaging the caged compound to transiently increase the ligand concentration in the focal region. The conformational change of protein caused by the interaction with the ligand is then monitored through visible pulse. Preliminary results clearly showed that the instrument developed here is applicable to analyze the ligand-induced conformational changes of proteins with 2D FLCS.
  Grant number：15K17814