SUPPLEMENTALDATA
ACOMPREHENSIVE MODEL FOR PACKING AND HYDRATION FOR AMYLOID FIBRILS OF 2-MICROGLOBULIN
Young-Ho Lee, EriChatani, Kenji Sasahara, Hironobu Naiki and Yuji Goto
Running head: Volumetric variationof amyloidogenesis
SUPPLEMENTALEXPERIMENTAL PROCEDURES
Mature fibrils ofK3 peptide- Lyophilized K3 monomer was first dissolved in 10 mMNaOH at 1 mMK3, and then, to trigger spontaneous fibrillation, the mixture was quickly diluted bya solution containing 2,2,2-trifluoroethanol (TFE), HCl, and NaCl as described (1). The final concentration of K3, HCl, NaCl, and TFE was adjusted to 100 M (0.25 mg·mL-1), 6 mM, 7 mM, and 20% (v/v), respectively. Thesample was incubated at25 °C for ~24 h.Density values of fibril solution containing 20% (v/v) TFE measured by a densitometer at 5 °C were irreproducible fluctuating highly within an order of magnitude of 1 10-3. Moreover, it was found that even a low percentage of TFE hampered the acquirement of reliable density values for the fibrils of K3 peptide, which took density values with an order of magnitude of 1 10-6 in our experimental system, due to the high intrinsic density and irreproducibility of density of TFEs. To exclude the effect of TFE on the density value of K3 fibrils, therefore, we carefully investigated the permissible range of TFE concentrations by measuring density for mixtures of water and TFE in various concentrations with a densitometer at 5 °C. We found that the density of the water and 0.00091% (v/v) TFE mixture was comparable with an experimental error of pure water in the densitymeasurements. To prepare K3 fibrils in a solution containing 0.00091% (v/v) TFE, 6 mM HCl, and 7 mM NaCl, we carried out consecutive seeding-dependent elongation experiments through repeated generation of K3 fibrils as follows; the first generation of K3 fibrils (1stMFK3, 20% TFE) spontaneously formed was used as the first seed (1stSeed) prepared by ultrasonicating 1stMFK3, and the final concentration of 1stSeed was adjusted to 1% (v/v) of total volume for the second generation of K3 fibrils (2ndMFK3, 0.2% TFE).Likewise, the second seed (2ndSeed, 0.2% TFE) was adjusted to 7% of the third generation of K3 fibrils (3rdMFK3, 0.014% TFE), and the third seed (3rdSeed, 0.014% TFE) was adjusted to 6.5% of the fourth generation of K3 fibrils (4thMFK3, 0.00091% TFE). Finally, 4thMFK3 was incubated at 25 °C for 90 h. Unless otherwise stated, fibrils of K3 peptide (MFK3) indicate the fourth generation of K3 fibrils (4thMFK3) throughout this study.
(1stSeed) (2ndSeed) (3rdSeed)
1% 7% 6.5% (Scheme 1)
1stMFK3 2ndMFK3 3rdMFK3 4thMFK3
(20% TFE) (0.2% TFE) (0.014% TFE) (0.00091% TFE)
It should be noted that the solution of each generation of K3 fibrils were transparent and stable in the presence of 7 mM NaCl even after several weeks. This implies prior to the sedimentation velocity analysis with analytical ultracentrifugationthat the solution including 6 mM HCl and 7 mM NaCl is highly suitable for densitometry because turbid solution of fibrils are apt to precipitate and impede accurate interpretations of density and/or volume.
Preparation of mature amyloid fibrils of A(1-40)- The mature fibrils of A(1-40) of 0.17 mg/ml were seed-dependently elongated overnight in a 50 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl at 37 C.The polymerization reaction of A(1-40) monomer was traced by fluorometricanalysis using thioflavin T, monitoredat 485 nm with an excitation at 445 nm with a Hitachi fluorescencespectrophotometer (data not shown) (2).
Polymerization and depolymerization of 2-m- Mature fibrils of 2-m were seed-dependently formed in a 50 mM citrate buffer (pH 2.5) containing 100 mM NaCl at 37 C.Immature fibrils of 2-m were spontaneously elongated in a 50 mM citrate buffer (pH 2.5) containing 400 mM NaCl at 37 C. The presence of both fibrils was confirmed by far-UV CD and AFM (data not shown).Gdn-HCl-induced unfolding (i.e., depolymerization) reactions of both fibrils were conducted in the same solutions where each fibril was formed except for Gdn-HCl concentrations. Both sample solutions were incubated overnight and the unfoldingof fibrils was monitored by examining the change in the CDspectrum at pH 2.5.The equilibriumunfolding curves were constructed by measuring the ellipticity at 220 nm for mature fibrils and at 215 nm for immature fibrils after incubation overnight at variousconcentrations of Gdn-HCl at pH 2.5 and 25 C.
CD measurements- Far-UV CD measurements of (im)mature fibrils of 2-m were performed at various concentrations of Gdn-HCl at 25C with a Jasco spectropolarimeter,model J600 using cells with a light path of 1 mm.
Sedimentation velocity measurement with analytical ultracentrifugation- The sedimentation velocity measurements were carried out on the mature amyloid fibrils of A(1-40) at 5 °C usinga Beckman-Coulter Optima XL-1 analytical ultracentrifuge (Fullerton,CA) equipped with an An-60 rotor and two- or six-channel charcoal-filled Eponcells.The sedimentation curves were recorded using absorbance data at 224 nm. The sedimentation measurements were conducted in a 50 mM sodium phosphate buffer of pH 7.2 containing 100 mM NaCl. Sincethe fibrils expeditiously sedimented during pre-centrifugation at 700 g for 5 minutes, further sedimentation velocity measurements were not performed.
Evaluation of sedimentation coefficient- Sedimentation velocity measurementsof the (short) mature and immature fibrils of 2-mas well as K3 fibrils were made usinga Beckman-Coulter Optima XL-1 analytical ultracentrifuge (Fullerton,CA) equipped with an An-60 rotor and two- or six-channel charcoal-filled Eponcells.The samples were centrifuged at 17,400 g and 5 °C, and the absorbance data at 280 nm were collected at intervals of 10-20 minutes. The sedimentation coefficients ofS20,w,which indicates the density and velocity of pure water at 20 °C, wereestimatedon the basis of the van Holde-Weischet method with the software UltraScan 8.0 ( (3).
Volume estimation of outside the core of fibrils- Simple estimation of corresponding to outside the core region in (im)mature fibrils was conducted based on Equation 1. We assumed that the core structure of K3 fibrils is accommodated at the center of (im)mature fibrils (Fig. 5B).
M(I)F2-m = MFK3 fK3OUT fOUT (Eq. S1)
where M(I)F2-m is the of mature or immature fibrils of 2-m,MFK3 is the value of K3 fibrils, and OUT is the of interest corresponding to the region outside of the core structure (K3 fibrils) in (im)mature fibrils. fK3 is the molecular weight ratio of K3 to 2-m, and fOUT is the molecular weight ratio of the sequences except K3 in 2-m to 2-m.
REFERENCES
1.Yamaguchi, K., Takahashi, S., Kawai, T., Naiki, H., and Goto, Y. (2005) J Mol Biol352, 952-960
2. Hasegawa, K., Yamaguchi,I.,Omata, S.,Gejyo, F.,and Naiki, H.(1999) Biochemistry38, 15514–15521
3.Demeler, B., and Saber, H. (1998) Biophys J74,444-454
Supplemental Fig.S1.Analytical ultracentrifugation experiments of fibrils.A.Sedimentation velocityprofile of mature amyloid fibrils of A(1-40). The sedimentation boundary profiles of the mature fibrils of A(1-40)were recorded by monitoring the absorbance at 224 nm at 700 g (3,000 rpm) and 5 °C. Initial absorbance before centrifugation was shown by closed circles at the left axis. B. Distribution of sedimentation coefficient (S20w)of the three different fibrils.The integral distribution of the mature fibrils of 2-m (●), the short mature fibrils of 2-m ultrasonicated (○), the immature fibrils of 2-m (▲), and the mature fibrils of K3 (■) was plotted as a function of S20w. Each distribution plot wasobtained by using the sedimentation velocity data measured at 17,400 g (15,000 rpm) and 5 °C.
Supplemental Fig.S2.Unfolding transitions by Gdn-HCl of the mature amyloid and immature amyloid-like fibrils of 2-m at pH 2.5.Depolymerization reactionsof the mature and immature fibrils of 2-m were monitored by CD at 220 nm (●) (A) and at 215 nm (▲) (B), respectively.
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