Supporting Information

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1 Supporting Information Copper and zinc ions specifically promote non-amyloid aggregation of the highly stable human γ-d crystallin Liliana Quintanar, 1,* José A. Domínguez-Calva, 1 Eugene Serebryany, 2 Lina Rivillas- Acevedo, 3 Cameron Haase-Pettingell, 2 Carlos Amero, 3 Jonathan A. King, 2,* 1 Departamento de Química, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, México 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 3 Centro de Investigaciones Químicas, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, México. jaking@mit.edu; lilianaq@cinvestav.mx Keywords: lens crystallins, human gamma-d crystallin, copper, zinc, cataracts

2 Recombinant HγD crystallin expression and purification. Recombinant HγD crystallin was expressed using a pet16b plasmid in BL21-RIL E. coli, and purified by ammonium sulfate precipitation followed by size exclusion chromatography (SEC). Briefly, this involves growing cells at 37 C in super broth to an optical density of 5 at 600 nm. For the production of 15 N and 13 C doubly labeled recombinant protein, the cells were grown in minimal M9 media containing 15 NH 4 Cl (1g/L, Cambridge Isotopes) and 13 C-glucose (2g/L). In all cases, protein expression was induced with 1 mm isopropyl β-d-1- thiogalactopyranoside (ITPG) at 18 C overnight. Harvested cells from a 2 L culture growth were resuspended with 10 mm ammonium acetate buffer ph 7, 50 mm NaCl, to a total volume of 50 ml. Cells were lysed by incubation with lysozyme (1 mg/ml) and DNase (20 ng/ml) for 30 min, in the presence of EDTA-free Complete-mini protease inhibitor cocktail (Roche), followed by sonication cycles (30 s each) in ice. Lysate was centrifuged at 17,000 g for 45 min, and ammonium sulfate was slowly added to the supernatant to reach 30% (w/v), while stirring in ice. The proteins that precipitated at 30 % ammonium sulfate were discarded, and more ammonium sulfate was added to reach 50% (w/v) and cause HγD crystallin precipitation. The precipitate was resuspended in 10 mm ammonium acetate buffer ph 7, with 50 mm NaCl, spun down, and passed through a 0.2 µm filter before loading into a SEC column (Hi-Prep Sephacryl S-100), using a fast protein liquid chromatography (FPLC) instrument (GE Life Sciences). Eluted HγD crystallin fractions were analyzed by SDS-PAGE to verify purity, pooled and stored at 4 C.

3 Thermal stability measurements by CD and fluorescence. Calculation of the fraction of unfolded protein. The fraction of unfolded (F u ) protein at each temperature was calculated according to Equation 1: F u = ( y-y f )/(y u -y) (Eq. 1) Where y is the CD intensity at 218 nm or the ratio of fluorescence intensity at 350 and 325 nm (FI 350/325) at a given temperature; y u is the intensity associated to the fully unfolded protein, and y N is the intensity associated to the native folded protein. Melting temperatures (T m ) were calculated by determining the midpoints of the thermal transitions where F u = 0.5, as previously described (Greenfield N. J. Nat. Protoc. 2006, 1, and Flaugh, S. L.; Mills, I. A.; King, J. J. Biol. Chem. 2006, 281, ).

4 Figure S1. Effect of temperature in the metal-induced aggregation of HγD crystallin. Turbidity assays of HγD crystallin (50 µm) in the presence of 3 equiv of Cu(II) (A), 10 equiv of Cu(II) (B), or 10 equiv of Zn(II) (C), at 27 C (light blue), 32 C (yellow), 37 C (orange), or 42 C (red). In all cases, the absorbance at 405 nm is reported as function of time after the addition of the metal ion.

5 Figure S2. Initial burst in turbidity is associated to protein aggregation. A rapid increase in turbidity is observed at early times when 10 equiv of Cu(II) are added to HγD crystallin (50 µm) at 37 C (blue trace in A); in contrast, no turbidity is observed when acetate buffer is incubated with 10 equiv of Cu(II) (black trace in A); in both cases, the relative absorbance at 405 nm is reported as function of time. Thus, initial increase in absorption intensity at 405 nm is not associated to Cu absorption, but to formation of protein aggregates. Consistently, the full absorption spectrum collected on the HγD crystallin (50 µm) sample 7 min after the addition of 10 equiv of Cu(II) shows increased turbidity at all wavelengths (blue trace in B). Upon centrifugation of the sample, the spectrum shows no turbidity, and the absorption at 280 nm indicates that only 30% of the protein remains soluble.

6 Figure S3. Thioflavin T assay for the HγD crystallin aggregates. ThT (50 µm) was added to the protein aggregates at the end point of the turbidity assay at 37 C for: HγD crystallin (50 µm) in the absence (grey) and presence of 2, 4, and 10 equiv of Cu(II) (blue traces), or 4 equiv of Zn(II) (red). The emission fluorescence spectrum was collected in each case, using an excitation wavelength of 450 nm, in a Cary Eclipse fluorimeter, and with a 1 cm path length quartz cell. ThT is a dye that fluoresces upon binding to amyloid fibrils. As a positive control, ThT (20 µm) was added to beta-amyloid fibrils that were formed by incubation of beta-amyloid(1-40) peptide (20 µm) in the absence (light green) or presence (dark green) of 1 equiv. of Cu(II) ions at 37 C; the amyloid nature of the beta-amyloid aggregates is revealed by the intense ThT fluorescence with a maximum intensity at 485 nm. In contrast, metal-induced aggregates of HγD crystallin do not bind ThT, and thus, do not cause significant ThT fluorescence; only a small baseline shift is observed at higher concentrations of copper (4 and 10 equiv), due to light scattering by the aggregates. Thus, the nature of the metal-induced aggregates of HγD crystallin is not amyloid.

7 Figure S4. Effect of oxygen in the copper-induced aggregation of HγD crystallin. A) Turbidity assays of HγD crystallin with 4 equiv of Cu(II) in the presence of oxygen (solid line) and under anaerobic conditions (dotted line). B) Aggregates obtained at the end of turbidity assays of HγD crystallin with 0 and 4 equiv of Cu(II) under normal atmospheric environment, and with 4 equiv of Cu(II) under anaerobic conditions.

8 Figure S5. Effect of Cu(II) ions in the folding of HγD crystallin by CD at 37 C. Titration of HγD crystallin (grey spectrum) with 1, 2, 3, 4, 5, 6, 8, and 10 equiv of Cu(II) (light to dark blue traces) was performed at a lower protein concentration (0.5 µm), to allow observation of the positive CD signal at 195 nm, that together with the 218 nm signal, is characteristic of β-sheet folding. Relative CD intensities at 218 and 195 nm are plotted as a function of Cu(II) added in the inset, showing a similar trend.

9 Figure S6. No aggregation occurs in the course of the CD folding experiment (Figure 4A). Titration of HγD crystallin (2 µm grey spectrum) with 1, 2, 3, 4, 5, 6, 8, and 10 equiv of Cu(II) (light to dark blue traces) was performed at 37 C, and followed by CD (A). Simultaneously, the voltage at the CD detector, which is proportional to the absorption of light, was monitored (B), showing no significant changes during the course of the experiment. If the protein were aggregating in the course of the experiment, the scattering of light would be evident in the CD detector voltage. Moreover, a UV-Vis absorption spectrum of the protein solution was collected at the beginning and at the end of the CD experiment, finding practically identical spectra (C). The absorbance at 280 nm is almost the same at the beginning and the end of the experiment, indicating that we are not losing soluble protein during the course of this experiment. Initial absorption at 280 nm corresponds to an initial protein concentration of 2.00 µm, while the final concentration was 1.98 µm.

10 Figure S7. Thermal denaturation curves for HγD crystallin in the presence of Cu(II) and Zn(II), followed by Trp fluorescence. Thermal denaturation of HγD crystallin (2 µm) was done in the presence of 0, 1, 2, 3, 4, or 5 equiv of Cu(II) (A) or Zn(II) (B). In all cases, the fraction of unfolded protein is plotted as a function of temperature. Data for at least duplicate experiments are shown, and the midpoints of the denaturation curves for each condition are listed in Table S1.

11 Table S1. Tm values for HγD crystallin in the presence of Cu(II) or Zn(II) ions. # equiv of Cu(II) Tm ( o C) Tm ( o C) by Fluorescence by CD ± ± ± ± ± ± ± ± ± ± ± ± 2.85 # equiv of Zn(II) Tm ( o C) by CD Tm ( o C) by Fluorescence ± ± ± ± ± ± ± ± ± ±2.41

12 Figure S8. 1H 15N-HSQC spectra of HγD crystallin 100 µm in the absence (black) or presence of 1.5 equivalents of Zn(II) (red) (A), and 1.5 equivalents of Cu(II) (B). The blue square correspond to the amplified region showed in figure 5.

13 Figure S9. 1 H 15 N-HSQC spectra of HγD crystallin 200 µm in the absence (black) or presence of 1.0 equivalent of Mn(II) (red) (A) and 1.0 equivalent of Ca(II) (red) (B). It should be noted that, due to technical issues (broken cryo-probe), this experiment had to be done at a protein concentration that is double than the one used in other NMR experiments, and the signals are broader than in previous experiments. Nevertheless, the signals associated to monomeric protein could be assigned, and they do not change upon addition of Ca(II) or Mn(II).