File S1 - Characterization of the Oligomerization and Aggregation of Human Serum Amyloid A

Figure S1, Monitoring TEV-assisted proteolysis of (His)6-TEV-hSAA1.1 to hSAA1.1 by SDS-PAGE: (lane 1), TEV protease; (lane 2), (His)6-TEV-hSAA1.1 obtained post-IMAC and SEC; (lane 3–8), TEV cleavage of (His)6-TEV-hSAA1.1 to generate hSAA1.1 monitored as a function of time; (lane 9) hSAA1.1 post-SEC purification. Figure S2, Influence of concentration on the oligomerization behavior of MetSAA1.1 and hSAA1.1: (A and B) SEC elution profiles of MetSAA1.1 and hSAA1.1 respectively refolded at 75 µM (black solid line), 20 µM (blue solid line), and 8.5 µM (red solid line) concentrations; (C and D) Far-UV CD-based thermal denaturation profiles of MetSAA1.1 and hSAA1.1 respectively at 20 µM (blue solid line) and 8.5 µM (green solid line) concentrations; (E and F) Tryptophan fluorescence emission spectra of MetSAA1.1 and hSAA1.1 respectively at 20 µM (blue solid line) and 8.5 µM (green solid line) concentrations. Figure S3, Characterization of residual structure of MetSAA1.1 and hSAA1.1 in urea solution: SEC elution profiles of (A) MetSAA1.1 (blue solid line) and (B) hSAA1.1 (red solid line) in 8 M urea solution; (C) Far-UV CD of MetSAA1.1 in 0 M (black solid line), 1 M (blue solid line), and 2 M (red solid line) urea solutions; (D) Far-UV CD of hSAA1.1 in 0 M (black solid line), 1 M (blue solid line), and 2 M (red solid line) urea solutions. Concentration of proteins used for all the assays was 20 µM. Assays were performed at 4°C. Figure S4, “Cross-purification” studies to analyze the oligomerization behavior of MetSAA1.1 and hSAA1.1: SEC elution profiles of (A) “regular” MetSAA1.1 (blue solid line) and “cross-purified” MetSAA1.1 (red solid line); (B) “regular” hSAA1.1 (blue solid line) and “cross-purified” hSAA1.1 (red solid line). Concentration of proteins used was 20 µM. Assays were performed at 4°C. Figure S5, Biophysical characterization of “native-like” oligomers formed by MetSAA1.1 and hSAA1.1: AFM analysis of (A) MetSAA1.1 “native-like” oligomers; (B) hSAA1.1 “native-like” oligomers. All scale bars for AFM images represent 1 µm. Figure S6, Biophysical characterization of aggregates formed by MetSAA1.1 and hSAA1.1: AFM analysis of (A) MetSAA1.1, 6 h, 37°C; (B) MetSAA1.1, 24 h, 37°C; (C) hSAA1.1, 12 h, 37°C; (D) hSAA1.1, 48 h, 37°C. All scale bars for AFM images represent 1 µm. Figure S7, Characterization of “cross-seeding” properties of MetSAA1.1 by ThT fluorescence assay: ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ MetSAA1.1 “seed” (gray bars. The concentration of protein was 20 µM. ThT fluorescence intensities were recorded by incubating the samples at 37°C. (DOC)