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This research was originally published in the Journal of Biological Chemistry. Masayuki Adachi, Masatomo So, Kazumasa Sakura, József Kardos and Yuji Goto. Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation. J. Biol. Chem. 2015; 290, 18134-18145. © the American Society for Biochemistry and Molecular Biology, Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.

This research was originally published in the Journal of Biological Chemistry. Yoko Akazawa-Ogawa, Mizuki Takashima, Young-Ho Lee, Takahisa Ikegami, Yuji Goto, Koichi Uegaki and Yoshihisa Hagihara. Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications. J. Biol. Chem. 2014; 289, 15666–15679. © the American Society for Biochemistry and Molecular Biology, The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

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, The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β2-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

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, To understand the mechanism by which amyloid fibrils form, we have been making real-time observations of the growth of individual fibrils, using total internal fluorescence microscopy combined with an amyloid-specific fluorescence dye, thioflavin T (ThT). At neutral pH, irradiation at 442 nm with a laser beam to excite ThT inhibited the fibril growth of β2-microglobulin (β2-m), a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Examination with a 22-residue K3 fragment of β2-m showed that the inhibition of fibril growth and moreover the destruction of preformed fibrils were coupled with the excitation of ThT. Several pieces of evidence suggest that the excited ThT transfers energy to ground state molecular oxygen, producing active oxygen, which causes various types of chemical modifications. The results imply a novel strategy for preventing the deposition of amyloid fibrils and for destroying preformed amyloid deposits.

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, Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid β. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of β2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mm HCl (pH ∼2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net π-π* transition moment of the backbone carbonyl group and Lb transition moment of the Tyr26 side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.

This research was originally published in the Journal of Biological Chemistry. Young-Ho Lee, Kosuke Tamura, Masahiro Maeda, Masaru Hoshino, Kazumasa Sakurai, Satoshi Takahashi, Takahisa Ikegami, Toshiharu Hase, and Yuji Goto. Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange. J. Biol. Chem. 2007; 282, 5959-5967. © the American Society for Biochemistry and Molecular Biology, NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP+ reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP+)-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pDr values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pDr values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP+-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP+-binding domain at pDr 8.0, the daytime pH in chloroplasts, than at pDr 6.0 is likely to be involved in the increased binding of NADP+ for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

This research was originally published in the Journal of Biological Chemistry. Yumiko Ohhashi, Kazuhiro Hasegawa, Hironobu Naiki and Yuji Goto. Optimum Amyloid Fibril Formation of a Peptide Fragment Suggests the Amyloidogenic Preference of β2-Microglobulin under Physiological Conditions. J. Biol. Chem. 2004; 279, 10814-10821. © the American Society for Biochemistry and Molecular Biology, β2-Microglobulin (β2m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length β2m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of β2m, Ser20–Lys41, obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length β2m. This suggests that once the rigid native-fold of β2m is unfolded and additional factors triggering the nucleation process are provided, full-length β2m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys25. The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length β2m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.

This research was originally published in the Journal of Biological Chemistry. Masanori Yagi, Kazumasa Sakurai, Chitkala Kalidas, Carl A. Batt and Yuji Goto. Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group. J. Biol. Chem. 2003; 278, 47009-47015. © the American Society for Biochemistry and Molecular Biology, Bovine β-lactoglobulin (β-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three β-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type β-lg. Urea-induced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 m urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type β-lg in 8 m urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type β-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of β-lg more exactly under various conditions.

This research was originally published in the Journal of Biological Chemistry. Tadato Ban, Daizo Hamada, Kazuhiro Hasegawa, Hironobu Naiki and Yuji Goto. Direct Observation of Amyloid Fibril Growth Monitored by Thioflavin T Fluorescence. J. Biol. Chem. 2003; 278, 16462-16465. © the American Society for Biochemistry and Molecular Biology, Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of β2-microgobulin (β2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent β2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid β-peptide.

This research was originally published in the Journal of Biological Chemistry. Dong-Pyo Hong, Masayo Gozu, Kazuhiro Hasegawa, Hironobu Naiki and Yuji Goto. Conformation of β2-Microglobulin Amyloid Fibrils Analyzed by Reduction of the Disulfide Bond. J. Biol. Chem. 2002; 277, 21554-21560. © the American Society for Biochemistry and Molecular Biology, β2-Microglobulin (β2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of β2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mmdithiothreitol. Because β2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.

This research was originally published in the Journal of Biological Chemistry. Gennady V. Kozhukh, Yoshihisa Hagihara, Toru Kawakami, Kazuhiro Hasegawa, Hironobu Naiki and Yuji Goto. Investigation of a Peptide Responsible for Amyloid Fibril Formation of β2-Microglobulin by Achromobacter Protease I. J. Biol. Chem. 2002; 277, 1310-1315. © the American Society for Biochemistry and Molecular Biology, To obtain insight into the mechanism of amyloid fibril formation from β2-microglobulin (β2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser20–Lys41 (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact β2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact β2-m seeds. Fibril formation of K3 peptide with intact β2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact β2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact β2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact β2-m, the K3 fibrils are less stable than the intact β2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.