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• W2885513945 abstract "Several serious diseases are associated with crystal-like amyloid fibrils or glass-like amorphous aggregates of denatured proteins. However, protein aggregation involving both types of aggregates has not yet been elucidated in much detail. Using a protein associated with dialysis-related amyloidosis, β2-microglobulin (β2m), we previously demonstrated that amyloid fibrils and amorphous aggregates form competitively depending on salt (NaCl) concentration. To examine the generality of the underlying competitive mechanisms, we herein investigated the effects of heat on acid-denatured β2m at pH 2. Using thioflavin fluorescence, CD, and light scattering analysis along with atomic force microscopy imaging, we found that the temperature-dependent aggregation of β2m markedly depends on NaCl concentration. Stepwise transitions from monomers to amyloids and then back to monomers were observed at low NaCl concentrations. Amorphous aggregates formed rapidly at ambient temperatures at high NaCl concentrations, but the transition from amorphous aggregates to amyloids occurred only as the temperature increased. Combining the data from the temperature- and NaCl-dependent transitions, we constructed a unified phase diagram of conformational states, indicating a parabolic solubility curve with a minimum NaCl concentration at ambient temperatures. Although amyloid fibrils formed above this solubility boundary, amorphous aggregates dominated in regions distant from this boundary. Kinetic competition between supersaturation-limited slow amyloid fibrillation and supersaturation-unlimited fast amorphous aggregation deformed the phase diagram, with amyloid regions disappearing with fast titration rates. We conclude that phase diagrams combining thermodynamics and kinetics data provide a comprehensive view of β2m aggregation exhibiting severe hysteresis depending on the heat- or salt-titration rates. Several serious diseases are associated with crystal-like amyloid fibrils or glass-like amorphous aggregates of denatured proteins. However, protein aggregation involving both types of aggregates has not yet been elucidated in much detail. Using a protein associated with dialysis-related amyloidosis, β2-microglobulin (β2m), we previously demonstrated that amyloid fibrils and amorphous aggregates form competitively depending on salt (NaCl) concentration. To examine the generality of the underlying competitive mechanisms, we herein investigated the effects of heat on acid-denatured β2m at pH 2. Using thioflavin fluorescence, CD, and light scattering analysis along with atomic force microscopy imaging, we found that the temperature-dependent aggregation of β2m markedly depends on NaCl concentration. Stepwise transitions from monomers to amyloids and then back to monomers were observed at low NaCl concentrations. Amorphous aggregates formed rapidly at ambient temperatures at high NaCl concentrations, but the transition from amorphous aggregates to amyloids occurred only as the temperature increased. Combining the data from the temperature- and NaCl-dependent transitions, we constructed a unified phase diagram of conformational states, indicating a parabolic solubility curve with a minimum NaCl concentration at ambient temperatures. Although amyloid fibrils formed above this solubility boundary, amorphous aggregates dominated in regions distant from this boundary. Kinetic competition between supersaturation-limited slow amyloid fibrillation and supersaturation-unlimited fast amorphous aggregation deformed the phase diagram, with amyloid regions disappearing with fast titration rates. We conclude that phase diagrams combining thermodynamics and kinetics data provide a comprehensive view of β2m aggregation exhibiting severe hysteresis depending on the heat- or salt-titration rates. There are more than 30 disease-related amyloidogenic proteins, including β-amyloid peptide associated with Alzheimer's disease and β2-microglobulin (β2m) 3The abbreviations used are: β2mβ2-microglobulinAFMatomic force microscopyDSCdifferential scanning calorimetryTEMtransmission electron microscopyThTthioflavin TUmonomeric unfolded stateNnative stateΔGU-NGibbs free energy change of foldingΔHU-Nenthalpy change of foldingΔSU-Nenthalpy change of foldingΔCpheat capacity change of unfolding. with dialysis-related amyloidosis (1Uversky V.N. Fink A.L. Conformational constraints for amyloid fibrillation: the importance of being unfolded.Biochim. Biophys. Acta. 2004; 1698 (10.1016/j.bbapap.2003.12.008 15134647): 131-153Crossref PubMed Scopus (917) Google Scholar2Chiti F. Dobson C.M. Protein misfolding, functional amyloid, and human disease.Annu. Rev. Biochem. 2006; 75 (10.1146/annurev.biochem.75.101304.123901 16756495): 333-366Crossref PubMed Scopus (5130) Google Scholar, 3Knowles T.P. Vendruscolo M. Dobson C.M. The amyloid state and its association with protein misfolding diseases.Nat. Rev. Mol. Cell Biol. 2014; 15 (10.1038/nrm3810 24854788): 384-396Crossref PubMed Scopus (1555) Google Scholar, 4Tycko R. Physical and structural basis for polymorphism in amyloid fibrils.Protein Sci. 2014; 23 (10.1002/pro.2544 25179159): 1528-1539Crossref PubMed Scopus (170) Google Scholar, 5Sipe J.D. Benson M.D. Buxbaum J.N. Ikeda S. Merlini G. Saraiva M.J. Westermark P. Nomenclature 2014: amyloid fibril proteins and clinical classification of the amyloidosis.Amyloid. 2014; 21 (10.3109/13506129.2014.964858 25263598): 221-224Crossref PubMed Scopus (370) Google Scholar6Riek R. Eisenberg D.S. The activities of amyloids from a structural perspective.Nature. 2016; 539 (10.1038/nature20416 27830791): 227-235Crossref PubMed Scopus (307) Google Scholar). Various amyloidogenic proteins form amyloid fibrils with the common properties of highly ordered cross-β structures stabilized by peptide hydrogen bonds (2Chiti F. Dobson C.M. Protein misfolding, functional amyloid, and human disease.Annu. Rev. Biochem. 2006; 75 (10.1146/annurev.biochem.75.101304.123901 16756495): 333-366Crossref PubMed Scopus (5130) Google Scholar, 3Knowles T.P. Vendruscolo M. Dobson C.M. The amyloid state and its association with protein misfolding diseases.Nat. Rev. Mol. Cell Biol. 2014; 15 (10.1038/nrm3810 24854788): 384-396Crossref PubMed Scopus (1555) Google Scholar, 6Riek R. Eisenberg D.S. The activities of amyloids from a structural perspective.Nature. 2016; 539 (10.1038/nature20416 27830791): 227-235Crossref PubMed Scopus (307) Google Scholar, 7Greenwald J. Riek R. Biology of amyloid: structure, function, and regulation.Structure. 2010; 18 (10.1016/j.str.2010.08.009 20947013): 1244-1260Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar8Eisenberg D. Jucker M. The amyloid state of proteins in human diseases.Cell. 2012; 148 (10.1016/j.cell.2012.02.022 22424229): 1188-1203Abstract Full Text Full Text PDF PubMed Scopus (1224) Google Scholar). The kinetics of the formation of amyloid fibrils are also common to various amyloidogenic proteins and may be divided into two steps: the nucleation and elongation phases (9Morris A.M. Watzky M.A. Finke R.G. Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature.Biochim. Biophys. Acta. 2009; 1794 (10.1016/j.bbapap.2008.10.016 19071235): 375-397Crossref PubMed Scopus (530) Google Scholar, 10Arosio P. Knowles T.P. Linse S. On the lag phase in amyloid fibril formation.Phys. Chem. Chem. Phys. 2015; 17 (10.1039/C4CP05563B 25719972): 7606-7618Crossref PubMed Google Scholar11Jarrett J.T. Lansbury Jr., P.T. Seeding “one-dimensional crystallization” of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie?.Cell. 1993; 73 (10.1016/0092-8674(93)90635-4 8513491): 1055-1058Abstract Full Text PDF PubMed Scopus (1919) Google Scholar). The nucleation phase, the duration of which is a lag time, is long because of a high free-energy barrier. Once amyloid nuclei form, the elongation of fibrils occurs rapidly. Seed-dependent elongation is another common property of amyloid fibril formation, in which the nucleation phase is shortened or avoided by the addition of seed fibrils. β2-microglobulin atomic force microscopy differential scanning calorimetry transmission electron microscopy thioflavin T monomeric unfolded state native state Gibbs free energy change of folding enthalpy change of folding enthalpy change of folding heat capacity change of unfolding. Amorphous aggregates are an alternative form of aggregated denatured proteins. Amorphous aggregates are also associated with diseases, such as cataracts (12Ecroyd H. Carver J.A. Crystallin proteins and amyloid fibrils.Cell Mol. Life Sci. 2009; 66 (10.1007/s00018-008-8327-4 18810322): 62-81Crossref PubMed Scopus (199) Google Scholar, 13Stranks S.D. Ecroyd H. Van Sluyter S. Waters E.J. Carver J.A. von Smekal L. Model for amorphous aggregation processes.Phys. Rev. E Stat. Nonlin. Soft Matter. Phys. 2009; 80 (10.1103/PhysRevE.80.051907 20365006): 051907Crossref PubMed Scopus (29) Google Scholar). The inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases (14Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.Nature. 2002; 416 (10.1038/416507a 11932737): 507-511Crossref PubMed Scopus (2154) Google Scholar). In practice, amorphous aggregates often contaminate the preparation of amyloid fibrils. Amorphous aggregate designation often accommodates the oligomers responsible for the cytotoxicity of amyloidogenic proteins (15Bemporad F. Chiti F. Protein misfolded oligomers: experimental approaches, mechanism of formation, and structure-toxicity relationships.Chem. Biol. 2012; 19 (10.1016/j.chembiol.2012.02.003 22444587): 315-327Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 16Miti T. Mulaj M. Schmit J.D. Muschol M. Stable, metastable, and kinetically trapped amyloid aggregate phases.Biomacromolecules. 2015; 16 (10.1021/bm501521r 25469942): 326-335Crossref PubMed Scopus (62) Google Scholar). Despite their potential medical importance, the role that amorphous aggregates play in amyloid fibrillation has yet to be clarified. When amyloid fibrils are regarded as crystal-like precipitates of denatured proteins, amorphous aggregates are presumed to be a glass-like state populated under conditions where the driving forces of precipitation are too strong to retain amyloid fibrils (17Yoshimura Y. Lin Y. Yagi H. Lee Y.H. Kitayama H. Sakurai K. So M. Ogi H. Naiki H. Goto Y. Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (10.1073/pnas.1208228109 22908252): 14446-14451Crossref PubMed Scopus (214) Google Scholar, 18So M. Hall D. Goto Y. Revisiting supersaturation as a factor determining amyloid fibrillation.Curr. Opin. Struct. Biol. 2016; 36 (10.1016/j.sbi.2015.11.009 26774801): 32-39Crossref PubMed Scopus (47) Google Scholar). This viewpoint may enable a more comprehensive understanding of aggregation in terms of solubility, supersaturation (or supercooling), and competition between the two types of aggregated states (17Yoshimura Y. Lin Y. Yagi H. Lee Y.H. Kitayama H. Sakurai K. So M. Ogi H. Naiki H. Goto Y. Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (10.1073/pnas.1208228109 22908252): 14446-14451Crossref PubMed Scopus (214) Google Scholar, 18So M. Hall D. Goto Y. Revisiting supersaturation as a factor determining amyloid fibrillation.Curr. Opin. Struct. Biol. 2016; 36 (10.1016/j.sbi.2015.11.009 26774801): 32-39Crossref PubMed Scopus (47) Google Scholar19Muta H. Lee Y.H. Kardos J. Lin Y. Yagi H. Goto Y. Supersaturation-limited amyloid fibrillation of insulin revealed by ultrasonication.J. Biol. Chem. 2014; 289 (10.1074/jbc.M114.566950 24847058): 18228-18238Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). By considering solubility and supersaturation, we previously showed with β2m that amyloid fibril formation and amorphous aggregation competed in a manner that depended on NaCl concentrations, thereby creating a competitive mechanism of aggregation (17Yoshimura Y. Lin Y. Yagi H. Lee Y.H. Kitayama H. Sakurai K. So M. Ogi H. Naiki H. Goto Y. Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (10.1073/pnas.1208228109 22908252): 14446-14451Crossref PubMed Scopus (214) Google Scholar, 20Adachi M. So M. Sakurai K. Kardos J. Goto Y. Supersaturation-limited and unlimited phase transitions compete to produce the pathway complexity in amyloid fibrillation.J. Biol. Chem. 2015; 290 (10.1074/jbc.M115.648139 26063798): 18134-18145Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We then examined the heparin-dependent amyloid formation of hen egg white lysozyme, a model amyloidogenic protein, revealing two distinct mechanisms of amyloid formation (21Nitani A. Muta H. Adachi M. So M. Sasahara K. Sakurai K. Chatani E. Naoe K. Ogi H. Hall D. Goto Y. Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation.J. Biol. Chem. 2017; 292 (10.1074/jbc.M117.813097 29101231): 21219-21230Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In both cases, amyloid fibril formation competed against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. To establish the generality of the competitive mechanism, we are now focusing on the effects of temperature. A large number of studies have investigated the effects of heat on the formation of amyloid fibrils. Otzen and co-workers (22Andersen C.B. Hicks M.R. Vetri V. Vandahl B. Rahbek-Nielsen H. Thogersen H. Thøgersen I.B. Enghild J.J. Serpell L.C. Rischel C. Otzen D.E. Glucagon fibril polymorphism reflects differences in protofilament backbone structure.J. Mol. Biol. 2010; 397 (10.1016/j.jmb.2010.02.012 20156459): 932-946Crossref PubMed Scopus (49) Google Scholar, 23Jeppesen M.D. Hein K. Nissen P. Westh P. Otzen D.E. A thermodynamic analysis of fibrillar polymorphism.Biophys. Chem. 2010; 149 (10.1016/j.bpc.2010.03.016 20435401): 40-46Crossref PubMed Scopus (26) Google Scholar) reported the stepwise heat-induced formation and degradation (i.e. dissociation or depolymerization) of glucagon amyloid fibrils. We also examined the effects of heat on the fibril formation of β2m and other proteins (24Sasahara K. Naiki H. Goto Y. Kinetically controlled thermal response of β2-microglobulin amyloid fibrils.J. Mol. Biol. 2005; 352 (10.1016/j.jmb.2005.07.033 16098535): 700-711Crossref PubMed Scopus (48) Google Scholar25Sasahara K. Naiki H. Goto Y. Exothermic effects observed upon heating of β2-microglobulin monomers in the presence of amyloid seeds.Biochemistry. 2006; 45 (10.1021/bi0606748 16846219): 8760-8769Crossref PubMed Scopus (21) Google Scholar, 26Sasahara K. Yagi H. Naiki H. Goto Y. Heat-induced conversion of β2-microglobulin and hen egg-white lysozyme into amyloid fibrils.J. Mol. Biol. 2007; 372 (10.1016/j.jmb.2007.06.088 17681531): 981-991Crossref PubMed Scopus (89) Google Scholar, 27Sasahara K. Yagi H. Naiki H. Goto Y. Heat-triggered conversion of protofibrils into mature amyloid fibrils of β2-microglobulin.Biochemistry. 2007; 46 (10.1021/bi602403v 17316024): 3286-3293Crossref PubMed Scopus (27) Google Scholar, 28Sasahara K. Yagi H. Sakai M. Naiki H. Goto Y. Amyloid nucleation triggered by agitation of β2-microglobulin under acidic and neutral pH conditions.Biochemistry. 2008; 47 (10.1021/bi701968g 18211100): 2650-2660Crossref PubMed Scopus (57) Google Scholar, 29Sasahara K. Yagi H. Naiki H. Goto Y. Thermal response with exothermic effects of β2-microglobulin amyloid fibrils and fibrillation.J. Mol. Biol. 2009; 389 (10.1016/j.jmb.2009.04.026 19379758): 584-594Crossref PubMed Scopus (15) Google Scholar30Kardos J. Micsonai A. Pál-Gábor H. Petrik É. Gráf L. Kovács J. Lee Y.H. Naiki H. Goto Y. Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils.Biochemistry. 2011; 50 (10.1021/bi2000017 21388222): 3211-3220Crossref PubMed Scopus (48) Google Scholar). Moreover, using isothermal titration calorimetry, we performed calorimetric measurements of β2m amyloid fibrillation and compared thermodynamic parameters with those of protein folding (31Kardos J. Yamamoto K. Hasegawa K. Naiki H. Goto Y. Direct measurement of the thermodynamic parameters of amyloid formation by isothermal titration calorimetry.J. Biol. Chem. 2004; 279 (10.1074/jbc.M409677200 15494406): 55308-55314Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 32Ikenoue T. Lee Y.H. Kardos J. Saiki M. Yagi H. Kawata Y. Goto Y. Cold denaturation of α-synuclein amyloid fibrils.Angew. Chem. Int. Ed. Engl. 2014; 53 (10.1002/anie.201403815 24920162): 7799-7804Crossref PubMed Scopus (64) Google Scholar33Ikenoue T. Lee Y.H. Kardos J. Yagi H. Ikegami T. Naiki H. Goto Y. Heat of supersaturation-limited amyloid burst directly monitored by isothermal titration calorimetry.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (10.1073/pnas.1322602111 24753579): 6654-6659Crossref PubMed Scopus (74) Google Scholar). The findings obtained showed that amyloid fibrils were in a thermodynamic state of unique calorimetric properties, amyloid fibrils degraded to monomers at higher temperatures, and low-temperature–induced degradation (i.e. cold denaturation) occurred for amyloid fibrils of α-synuclein (32Ikenoue T. Lee Y.H. Kardos J. Saiki M. Yagi H. Kawata Y. Goto Y. Cold denaturation of α-synuclein amyloid fibrils.Angew. Chem. Int. Ed. Engl. 2014; 53 (10.1002/anie.201403815 24920162): 7799-7804Crossref PubMed Scopus (64) Google Scholar). However, the relationship between amyloid fibrils and amorphous aggregates with respect to temperature has not yet been elucidated. By combining a series of temperature- and salt-dependent transitions, we constructed a unified phase diagram of the conformational states of β2m. The phase diagram showed that phase transitions from soluble monomers to crystal-like amyloid fibrils and then glass-like amorphous aggregates are common to three types of variables that decrease the solubility of β2m (i.e. increasing NaCl concentrations, increasing the temperature at the low temperature region, or decreasing the temperature at the high temperature region), thereby providing a comprehensive view of protein aggregation. We investigated the dependence of β2m aggregation on temperature, for which the heating rate was controlled using a Peltier element (Fig. 1A and Figs. S1 and S2). Amyloid fibrillation was measured by thioflavin T (ThT) fluorescence at 485 nm with a heating rate of 0.2 °C/min. We simultaneously measured light scattering at 445 nm to monitor the total amount of aggregates. Based on these measurements, we distinguished amyloid fibrils and amorphous aggregates. Furthermore, to obtain information on secondary structures, CD measurements were performed by removing aliquots of the solution from the fluorometer at the desired time points. Under standard solvent conditions (8.5 μm (0.1 mg/ml) β2m, 0.1 m NaCl, 5 μm ThT, and 10 mm HCl under stirring) at a constant temperature of 25 °C, amyloid fibrillation occurred with a lag time of ∼3 h and finished at ∼5 h (Fig. 1, A and B). Increases in ThT and light scattering intensity occurred simultaneously, and the change in ellipticity was consistent with those in ThT and light scattering intensities (Fig. 1, B and C). These results indicated that the formation of amyloid fibrils occurred cooperatively without the accumulation of amorphous aggregates. When β2m solution in the absence of NaCl was heated at 0.2 °C/min, no aggregation occurred when monitored by ThT or light scattering (Fig. 1D). CD spectra showed that β2m remained unfolded (Fig. 1, E and F). Upon heating in the presence of 0.1 m NaCl, ThT and light scattering intensities both increased at ∼40 °C (Fig. 1G), indicating the formation of amyloid fibrils. ThT fluorescence significantly decreased with an increase in temperature. This decrease initially suggested the degradation of amyloid fibrils. However, because subsequent cycles of decreases and increases in temperature reproduced the temperature-dependent change in ThT fluorescence without affecting light scattering and the CD spectrum typical for the β-structure (Fig. S1), the decrease observed in ThT intensity was attributed to a reduction in the efficiency of the fluorescence of ThT at a high temperature. We previously showed with differential scanning calorimetry (DSC) that β2m amyloid fibrils at 0.1 mg/ml were completely degraded upon heating and that the transition temperature of degradation increased with elevations in the concentration of NaCl (see Fig. 4 of Ref. (24Sasahara K. Naiki H. Goto Y. Kinetically controlled thermal response of β2-microglobulin amyloid fibrils.J. Mol. Biol. 2005; 352 (10.1016/j.jmb.2005.07.033 16098535): 700-711Crossref PubMed Scopus (48) Google Scholar; see also Figure 2., Figure 5.). We also reported the complete degradation of β2m amyloid fibrils at 0.1–0.3 mg/ml upon a 10-min incubation at 99 °C monitored by ThT fluorescence and CD spectroscopy (30Kardos J. Micsonai A. Pál-Gábor H. Petrik É. Gráf L. Kovács J. Lee Y.H. Naiki H. Goto Y. Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils.Biochemistry. 2011; 50 (10.1021/bi2000017 21388222): 3211-3220Crossref PubMed Scopus (48) Google Scholar). Heat-induced degradation was also reported for various amyloid fibrils (32Ikenoue T. Lee Y.H. Kardos J. Saiki M. Yagi H. Kawata Y. Goto Y. Cold denaturation of α-synuclein amyloid fibrils.Angew. Chem. Int. Ed. Engl. 2014; 53 (10.1002/anie.201403815 24920162): 7799-7804Crossref PubMed Scopus (64) Google Scholar, 34Surmacz-Chwedoruk W. Malka I. Bozycki L. Nieznańska H. Dzwolak W. On the heat stability of amyloid-based biological activity: insights from thermal degradation of insulin fibrils.PLoS One. 2014; 9 (10.1371/journal.pone.0086320 24466022): e86320Crossref PubMed Scopus (18) Google Scholar, 35Pedersen J.S. The nature of amyloid-like glucagon fibrils.J. Diabetes Sci. Technol. 2010; 4 (10.1177/193229681000400609 21129330): 1357-1367Crossref PubMed Scopus (56) Google Scholar). The ellipticity value at 200 nm indicative of the β-structure component (Fig. 1, H and I) and the large amount of amyloid fibrils observed in atomic force microscopy (AFM) images taken after cooling (Fig. 1G, inset) suggested that the highest temperature employed under the current experimental conditions (90 °C; Fig. 1, G and H) was not sufficient to completely degrade amyloid fibrils.Figure 2.Degradation of β2m amyloid fibrils by heating. A, temperature-dependent degradation curves of amyloid fibrils that were formed in the presence of 100 (red), 75 (green), or 50 (blue) mm NaCl in 10 mm HCl at 37 °C. Normalized curves are shown. B–D, CD spectra at 25 °C (blue) and 100 °C (red) and that of the acid-denatured state (dashed line). E and F, AFM images of amyloid fibrils prepared in 100 mm NaCl (E) and those after heating to 100 °C (F). Scale bars represent 1 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5.Phase transitions of β2m dependent on temperature at various NaCl concentrations. A, transition boundary from amorphous aggregates (red) to amyloid fibrils (blue) at high NaCl concentrations obtained from changes in ThT and light scattering shown in Fig. 3. B, amyloid fibril region (blue) defined by temperature-dependent amyloid fibril formation or degradation at the low NaCl concentration region. Amorphous aggregates in this region (red) may be converted to amyloid fibrils in a prolonged incubation. Transition temperatures obtained from Refs. 24Sasahara K. Naiki H. Goto Y. Kinetically controlled thermal response of β2-microglobulin amyloid fibrils.J. Mol. Biol. 2005; 352 (10.1016/j.jmb.2005.07.033 16098535): 700-711Crossref PubMed Scopus (48) Google Scholar, 29Sasahara K. Yagi H. Naiki H. Goto Y. Thermal response with exothermic effects of β2-microglobulin amyloid fibrils and fibrillation.J. Mol. Biol. 2009; 389 (10.1016/j.jmb.2009.04.026 19379758): 584-594Crossref PubMed Scopus (15) Google Scholar, and 30Kardos J. Micsonai A. Pál-Gábor H. Petrik É. Gráf L. Kovács J. Lee Y.H. Naiki H. Goto Y. Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils.Biochemistry. 2011; 50 (10.1021/bi2000017 21388222): 3211-3220Crossref PubMed Scopus (48) Google Scholar are also included. Transition boundaries were drawn manually based on the observed data points without fitting and were also used for the schematic phase diagrams shown in Figure 6., Figure 7..View Large Image Figure ViewerDownload Hi-res image Download (PPT) When β2m solution in 1.0 m NaCl at 25 °C was prepared, amorphous aggregation rapidly occurred and was accompanied by an increase in light scattering without any elevations in ThT fluorescence, which is consistent with our previous findings (17Yoshimura Y. Lin Y. Yagi H. Lee Y.H. Kitayama H. Sakurai K. So M. Ogi H. Naiki H. Goto Y. Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (10.1073/pnas.1208228109 22908252): 14446-14451Crossref PubMed Scopus (214) Google Scholar, 20Adachi M. So M. Sakurai K. Kardos J. Goto Y. Supersaturation-limited and unlimited phase transitions compete to produce the pathway complexity in amyloid fibrillation.J. Biol. Chem. 2015; 290 (10.1074/jbc.M115.648139 26063798): 18134-18145Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) (Fig. 1J). Upon increases in temperature, light scattering gradually decreased. A sharp increase in ThT fluorescence occurred beginning at ∼45 °C. These results suggested that amyloid fibrils formed and were accompanied by the dissolution of amorphous aggregates. CD measurements also indicated the formation of amyloid fibrils at high temperatures, although these measurements were disturbed by contaminated amorphous aggregates (Fig. 1, K and L). AFM images showed coexisting amyloid fibrils and amorphous-like structures (Fig. 1J, inset). We previously reported the heat-induced conversion of protofibrils to rigid amyloid fibrils in 0.5 m NaCl monitored by DSC, CD, and transmission EM (TEM) (27Sasahara K. Yagi H. Naiki H. Goto Y. Heat-triggered conversion of protofibrils into mature amyloid fibrils of β2-microglobulin.Biochemistry. 2007; 46 (10.1021/bi602403v 17316024): 3286-3293Crossref PubMed Scopus (27) Google Scholar). In the present study, we assumed that salt-induced protofibrils were categorized into amorphous aggregates and that the heat-induced conversion observed was the same transition as that in our previous study (27Sasahara K. Yagi H. Naiki H. Goto Y. Heat-triggered conversion of protofibrils into mature amyloid fibrils of β2-microglobulin.Biochemistry. 2007; 46 (10.1021/bi602403v 17316024): 3286-3293Crossref PubMed Scopus (27) Google Scholar). To investigate the temperature-dependent stability of amyloid fibrils, amyloid fibrils were prepared under different NaCl concentrations at 25 °C (e.g. Fig. 1B, in 0.1 m NaCl), heated at 0.5 °C/min, and monitored by ellipticity at 220 nm (Fig. 2A). CD measurements confirmed the degradation of preformed amyloid fibrils at a specific temperature and that the midpoint of degradation increased at higher salt concentrations (Fig. 2A), which is consistent with previous findings (27Sasahara K. Yagi H. Naiki H. Goto Y. Heat-triggered conversion of protofibrils into mature amyloid fibrils of β2-microglobulin.Biochemistry. 2007; 46 (10.1021/bi602403v 17316024): 3286-3293Crossref PubMed Scopus (27) Google Scholar). Furthermore, AFM images showed that the fibrils observed before heating (Fig. 2E) disappeared after heating (Fig. 2F). CD spectra also showed the degradation of preformed amyloid fibrils at 100 °C (Fig. 2, B–D). Collectively, these results showed that amyloid fibrils were degraded at high temperature and that the degradation temperature increased with elevations in the salt concentration. Amyloid fibrillation was not observed at NaCl concentrations lower than 50 mm, e.g. 25 mm NaCl, even after an incubation at 25 °C for ∼12 h (data not shown); however, fibril formation occurred at higher temperatures. On the other hand, NaCl promoted the formation of amyloid fibrils with an increase in its concentration. Taken together, amyloid fibrils formed at moderate NaCl concentrations and at ambient temperature degraded at low and high temperatures. In other words, we observed the “cold denaturation” and “heat denaturation” of β2m amyloid fibrils, as evidently observed for α-synuclein amyloid fibrils (32Ikenoue T. Lee Y.H. Kardos J. Saiki M. Yagi H. Kawata Y. Goto Y. Cold denaturation of α-synuclein amyloid fibrils.Angew. Chem. Int. Ed. Engl. 2014; 53 (10.1002/anie.201403815 24920162): 7799-7804Crossref PubMed Scopus (64) Google Scholar). We previously reported that amorphous aggregates formed in the presence of high salt concentrations at 37 °C is a thermodynamic state stabilized by the salting-out effect (20Adachi M. So M. Sakurai K. Kardos J. Goto Y. Supersaturation-limited and unlimited phase transitions compete to produce the pathway complexity in amyloid fibrillation.J. Biol. Chem. 2015; 290 (10.1074/jbc.M115.648139 26063798): 18134-18145Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). I" @default.
• W2885513945 created "2018-08-22" @default.
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• W2885513945 date "2018-09-01" @default.
• W2885513945 modified "2023-10-17" @default.
• W2885513945 title "Aggregation-phase diagrams of β2-microglobulin reveal temperature and salt effects on competitive formation of amyloids versus amorphous aggregates" @default.
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