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Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications., identifier:https://www.sciencedirect.com/science/article/pii/S0021925820340461

Kichitaro Nakajima, Hirotsugu Ogi, Kanta Adachi, Kentaro Noi, Masahiko Hirao, Hisashi Yagi & Yuji Goto "Nucleus factory on cavitation bubble for amyloid β fibril", Scientific Reports, volume 6, Article number: 22015, Springer Nature, 2016

This research was originally published in the Journal of Biological Chemistry. Mayu S. Terakawa, Hisashi Yagi, Masayuki Adachi, Young-Ho Lee and Yuji Goto. Small Liposomes Accelerate the Fibrillation of Amyloid β (1–40). J. Biol. Chem. 2015; 290, 815-826. © the American Society for Biochemistry and Molecular Biology

Hiroki Hamada, Hirotsugu Ogi, Kentaro Noi, Hisashi Yagi, Yuji Goto and Masahiko Hirao. Nucleation-fibrillation dynamics of Aβ₁₋₄₀ peptides on liquid-solid surface studied by total-internal-reflection fluorescence microscopy coupled with quartz-crystal microbalance biosensor. Japanese Journal of Applied Physics, 2015, 54(7S1), 07HE01. https://doi.org/10.7567/JJAP.54.07HE01.

This research was originally published in the Journal of Biological Chemistry. Cardine N. Nokwe, Martin Zacharias, Hisashi Yagi, Manuel Hora, Bernd Reif, Yuji Goto and Johannes Buchner. A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains. J. Biol. Chem. 2014; 289, 26829-26846. © the American Society for Biochemistry and Molecular Biology

This research was originally published in the Journal of Biological Chemistry. Hiroya Muta, Young-Ho Lee, József Kardos, Yuxi Lin, Hisashi Yagi and Yuji Goto. Supersaturation-limited Amyloid Fibrillation of Insulin Revealed by Ultrasonication. J. Biol. Chem. 2014; 289, 18228–18238. © the American Society for Biochemistry and Molecular Biology

Hirotsugu Ogi, Masahiko Fukukshima, Hiroki Hamada, Kentaro Noi, Masahiko Hirao, Hisashi Yagi & Yuji Goto "Ultrafast propagation of β-amyloid fibrils in oligomeric cloud", Scientific Reports, volume 4, Article number: 6960, Springer Nature, 2014

This research was originally published in the Journal of Biological Chemistry. P. Patrizia Mangione, Gennaro Esposito, Annalisa Relini, Sara Raimondi, Riccardo Porcari, Sofia Giorgetti, Alessandra Corazza, Federico Fogolari, Amanda Penco, Yuji Goto, Young-Ho Lee, Hisashi Yagi, Ciro Cecconi, Mohsin M. Naqvi, Julian D. Gillmore, Philip N. Hawkins, Fabrizio Chiti, Ranieri Rolandi, Graham W. Taylor, Mark B. Pepys, Monica Stoppini and Vittorio Bellotti. Structure, Folding Dynamics, and Amyloidogenesis of D76N β2-Microglobulin: ROLES OF SHEAR FLOW, HYDROPHOBIC SURFACES, AND α-CRYSTALLIN. J. Biol. Chem. 2013; 288, 30917–30930. © the American Society for Biochemistry and Molecular Biology

This research was originally published in the Journal of Biological Chemistry. Yusuke Kato, Hisashi Yagi, Yuichi Kaji, Tetsuro Oshika and Yuji Goto. Benzalkonium Chloride Accelerates the Formation of the Amyloid Fibrils of Corneal Dystrophy-associated Peptides. J. Biol. Chem. 2013; 288, 25109-25118. © the American Society for Biochemistry and Molecular Biology

Kentaro Uesugi, Hirotsugu Ogi, Masahiko Fukushima, Masatomo So, Hisashi Yagi, Yuji Goto and Masahiko Hirao. Mechanisms of ultrasonically induced fibrillation of amyloid β₁₋₄₀ peptides. Japanese Journal of Applied Physics, 52(7S), 07HE10. https://doi.org/10.7567/JJAP.52.07HE10.

This research was originally published in the Journal of Biological Chemistry. Eri Chatani, Hisashi Yagi, Hironobu Naiki and Yuji Goto. Polymorphism of β2-Microglobulin Amyloid Fibrils Manifested by Ultrasonication-enhanced Fibril Formation in Trifluoroethanol. J. Biol. Chem. 2012; 287, 22827-22837. © the American Society for Biochemistry and Molecular Biology

This research was originally published in the Journal of Biological Chemistry. Daisaku Ozawa, Yuichi Kaji, Hisashi Yagi, Kazumasa Sakurai, Toru Kawakami, Hironobu Naiki, and Yuji Goto. Destruction of Amyloid Fibrils of Keratoepithelin Peptides byLaser Irradiation Coupled with Amyloid-specific Thioflavin T. J. Biol. Chem. 2011; 286, 10856-10863. © the American Society for Biochemistry and Molecular Biology

This research was originally published in the Journal of Biological Chemistry. Kotaro Yanagi, Mizue Ashizaki, Hisashi Yagi, Kazumasa Sakurai, Young-Ho Lee and Yuji Goto. Hexafluoroisopropanol Induces Amyloid Fibrils of Islet Amyloid Polypeptide by Enhancing Both Hydrophobic and Electrostatic Interactions. J. Biol. Chem. 2011; 286, 23959-23966. © the American Society for Biochemistry and Molecular Biology

Ogi H., Fukunishi Y., Yanagida T., et al. "Seed-dependent deposition behavior of Aβ peptides studied with wireless quartz-crystal-microbalance biosensor", Analytical Chemistry, 83(12), 4982-4988, May 11, 2011. Copyright © 2011 American Chemical Society. https://doi.org/10.1021/ac2007703.

This research was originally published in the Journal of Biological Chemistry. Hisashi Yagi, Daisaku Ozawa, Kazumasa Sakurai, Toru Kawakami, Hiroki Kuyama, Osamu Nishimura,Toshinori Shimanouchi, Ryoichi Kuboi, Hironobu Naiki, and Yuji Goto. Laser-induced Propagation and Destruction of Amyloid β Fibrils. J. Biol. Chem. 2010; 285, 19660-19667. © the American Society for Biochemistry and Molecular Biology

Many human diseases are associated with protein aggregation and fibrillation. We present experiments on in vitro glucagon fibrillation using total internal reflection fluorescence microscopy, providing real-time measurements of single-fibril growth. We find that amyloid fibrils grow in an intermittent fashion, with periods of growth followed by long pauses. The observed exponential distributions of stop and growth times support a Markovian model, in which fibrils shift between the two states with specific rates. Even if the individual rates vary considerably, we observe that the probability of being in the growing (stopping) state is very close to 1/4 (3/4) in all experiments.

Many human diseases are associated with protein aggregation and fibrillation. We present experiments on in vitro glucagon fibrillation using total internal reflection fluorescence microscopy, providing real-time measurements of single-fibril growth. We find that amyloid fibrils grow in an intermittent fashion, with periods of growth followed by long pauses. The observed exponential distributions of stop and growth times support a Markovian model, in which fibrils shift between the two states with specific rates. Even if the individual rates vary considerably, we observe that the probability of being in the growing (stopping) state is very close to 1/4 (3/4) in all experiments.

This research was originally published in the Journal of Biological Chemistry. Daisaku Ozawa, Hisashi Yagi, Tadato Ban, Atsushi Kameda, Toru Kawakami, Hironobu Naiki and Yuji Goto. Destruction of Amyloid Fibrils of a β2-Microglobulin Fragment by Laser Beam Irradiation. J. Biol. Chem. 2009; 284, 1009-1017. © the American Society for Biochemistry and Molecular Biology

This research was originally published in the Journal of Biological Chemistry. Rumi Adachi, Kei-ichi Yamaguchi, Hisashi Yagi, Kazumasa Sakurai, Hironobu Naiki and Yuji Goto. Flow-induced Alignment of Amyloid Protofilaments Revealed by Linear Dichroism. J. Biol. Chem. 2007; 282, 8978-8983. © the American Society for Biochemistry and Molecular Biology

Jin Chen, Hisashi Yagi, Yuji Furutani, Takashi Nakamura, Asumi Inaguma, Hao Guo, Yan Kong & Yuji Goto "Self-assembly of the chaperonin GroEL nanocage induced at submicellar detergent", Scientific Reports, volume 4, Article number: 5614, Springer Nature, 2014, Protein nanoassemblies possess unique advantage in biomedical applications such as drug delivery, biocatalysis and vaccine development. Despite recent accomplishment in atomic structure data, the underlying molecular mechanism of protein self-assembly remains elusive, where considerable heterogeneity is often involved. Here we use E. coli chaperonin GroEL, a tetradecameric protein with a molecular weight of 805 kDa, to probe its transformation from cage-like oligomers to protein nanofibers. We show that sodium dodecyl sulfate (SDS), a widely-used protein denaturant, at submicellar concentration binds to and causes partial distortion of GroEL apical domain. Subsequently, the GroEL apical domain with altered secondary structural content converts the GroEL oligomers into modular structural units which are observed to self-assemble into cylindrical nanofibers under an agitated incubation in a physiological buffer. Interestingly, through targeted mutagenesis where two cysteine residues are introduced at the entry site of GroEL cage, we found that the formation of GroEL nanoassembly could be modulated depending on the redox condition of incubation. Without the need of chemical engineering, tunable GroEL nanofibers built by controlled-assembly are among the largest nanoscale bioassembly with broad applications.

Jesper Ferkinghoff-Borg, Jesper Fonslet, Christian Beyschau Andersen, Sandeep Krishna, Simone Pigolotti, Hisashi Yagi, Yuji Goto, Daniel Otzen, and Mogens H. Jensen "Stop-and-go kinetics in amyloid fibrillation", Physical Review E, 82, 010901, American Physical Society, 2010, Many human diseases are associated with protein aggregation and fibrillation. We present experiments on in vitro glucagon fibrillation using total internal reflection fluorescence microscopy, providing real-time measurements of single-fibril growth. We find that amyloid fibrils grow in an intermittent fashion, with periods of growth followed by long pauses. The observed exponential distributions of stop and growth times support a Markovian model, in which fibrils shift between the two states with specific rates. Even if the individual rates vary considerably, we observe that the probability of being in the growing (stopping) state is very close to 1/4 (3/4) in all experiments.

This research was originally published in the Journal of Biological Chemistry. Ayaka Umemoto, Hisashi Yagi, Masatomo So and Yuji Goto. High-throughput Analysis of Ultrasonication-forced Amyloid Fibrillation Reveals the Mechanism Underlying the Large Fluctuation in the Lag Time. J. Biol. Chem. 2014; 289, 27290–27299. © the American Society for Biochemistry and Molecular Biology, Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism characterized by a lag time. Although the lag time provides a clue to understanding the complexity of nucleation events, its long period and low reproducibility have been obstacles for exact analysis. Ultrasonication is known to effectively break supersaturation and force fibrillation. By constructing a Handai amyloid burst inducer, which combines a water bath-type ultrasonicator and a microplate reader, we examined the ultrasonication-forced fibrillation of several proteins, with a focus on the fluctuation in the lag time. Amyloid fibrillation of hen egg white lysozyme was examined at pH 2.0 in the presence of 1.0–5.0 M guanidine hydrochloride (GdnHCl), in which the dominant species varied from the native to denatured conformations. Although fibrillation occurred at various concentrations of GdnHCl, the lag time varied largely, with a minimum being observed at ∼3.0 m, the concentration at which GdnHCl-dependent denaturation ended. The coefficient of variation of the lag time did not depend significantly on the GdnHCl concentration and was 2-fold larger than that of the ultrasonication-dependent oxidation of iodide, a simple model reaction. These results suggest that the large fluctuation observed in the lag time for amyloid fibrillation originated from a process associated with a common amyloidogenic intermediate, which may have been a relatively compact denatured conformation. We also suggest that the Handai amyloid burst inducer system will be useful for studying the mechanism of crystallization of proteins because proteins form crystals by the same mechanism as amyloid fibrils under supersaturation.

This research was originally published in the Journal of Biological Chemistry. Ayaka Umemoto, Hisashi Yagi, Masatomo So and Yuji Goto. High-throughput Analysis of Ultrasonication-forced Amyloid Fibrillation Reveals the Mechanism Underlying the Large Fluctuation in the Lag Time. J. Biol. Chem. 2014; 289, 27290–27299. © the American Society for Biochemistry and Molecular Biology, Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism characterized by a lag time. Although the lag time provides a clue to understanding the complexity of nucleation events, its long period and low reproducibility have been obstacles for exact analysis. Ultrasonication is known to effectively break supersaturation and force fibrillation. By constructing a Handai amyloid burst inducer, which combines a water bath-type ultrasonicator and a microplate reader, we examined the ultrasonication-forced fibrillation of several proteins, with a focus on the fluctuation in the lag time. Amyloid fibrillation of hen egg white lysozyme was examined at pH 2.0 in the presence of 1.0–5.0 M guanidine hydrochloride (GdnHCl), in which the dominant species varied from the native to denatured conformations. Although fibrillation occurred at various concentrations of GdnHCl, the lag time varied largely, with a minimum being observed at ∼3.0 m, the concentration at which GdnHCl-dependent denaturation ended. The coefficient of variation of the lag time did not depend significantly on the GdnHCl concentration and was 2-fold larger than that of the ultrasonication-dependent oxidation of iodide, a simple model reaction. These results suggest that the large fluctuation observed in the lag time for amyloid fibrillation originated from a process associated with a common amyloidogenic intermediate, which may have been a relatively compact denatured conformation. We also suggest that the Handai amyloid burst inducer system will be useful for studying the mechanism of crystallization of proteins because proteins form crystals by the same mechanism as amyloid fibrils under supersaturation.

Jesper Ferkinghoff-Borg, Jesper Fonslet, Christian Beyschau Andersen, Sandeep Krishna, Simone Pigolotti, Hisashi Yagi, Yuji Goto, Daniel Otzen, and Mogens H. Jensen "Stop-and-go kinetics in amyloid fibrillation", Physical Review E, 82, 010901, American Physical Society, 2010, Many human diseases are associated with protein aggregation and fibrillation. We present experiments on in vitro glucagon fibrillation using total internal reflection fluorescence microscopy, providing real-time measurements of single-fibril growth. We find that amyloid fibrils grow in an intermittent fashion, with periods of growth followed by long pauses. The observed exponential distributions of stop and growth times support a Markovian model, in which fibrils shift between the two states with specific rates. Even if the individual rates vary considerably, we observe that the probability of being in the growing (stopping) state is very close to 1/4 (3/4) in all experiments.

Jin Chen, Hisashi Yagi, Yuji Furutani, Takashi Nakamura, Asumi Inaguma, Hao Guo, Yan Kong & Yuji Goto "Self-assembly of the chaperonin GroEL nanocage induced at submicellar detergent", Scientific Reports, volume 4, Article number: 5614, Springer Nature, 2014, Protein nanoassemblies possess unique advantage in biomedical applications such as drug delivery, biocatalysis and vaccine development. Despite recent accomplishment in atomic structure data, the underlying molecular mechanism of protein self-assembly remains elusive, where considerable heterogeneity is often involved. Here we use E. coli chaperonin GroEL, a tetradecameric protein with a molecular weight of 805 kDa, to probe its transformation from cage-like oligomers to protein nanofibers. We show that sodium dodecyl sulfate (SDS), a widely-used protein denaturant, at submicellar concentration binds to and causes partial distortion of GroEL apical domain. Subsequently, the GroEL apical domain with altered secondary structural content converts the GroEL oligomers into modular structural units which are observed to self-assemble into cylindrical nanofibers under an agitated incubation in a physiological buffer. Interestingly, through targeted mutagenesis where two cysteine residues are introduced at the entry site of GroEL cage, we found that the formation of GroEL nanoassembly could be modulated depending on the redox condition of incubation. Without the need of chemical engineering, tunable GroEL nanofibers built by controlled-assembly are among the largest nanoscale bioassembly with broad applications.