Myostatin Amyloid Formation Figure 2. Refolding and purification of human MstnPP. A. Reducing (+ b-mercaptoethanol (bME)) vs non-reducing (2bME) SDS-PAGE (12%) after refolding. b-ME concentration is 2 M. Bands are as indicated: 1. soluble aggregates; 2. disulfide-bonded dimer; 3. monomer either fully reduced (+b-ME) or misfolded at various stages of reduction (2b-ME). Figure assembled from one gel where intervening lanes were removed. B. Heparin affinity chromatography separates the majority of soluble aggregates (H2) from correctly-folded dimer (H1). C. S200 gel filtration chromatography; the MstnPP dimer elutes at 13.5 mL (PD) and the soluble aggregates in the void volume at 8 mL (PA). doi:10.1371/journal.pone.0009170.g002 first structural analysis for any TGF-b family precursor protein. Quite a different profile is observed for the MstnPP soluble aggregates with the very strong minimum at 208 nm indicating that the secondary structure is primarily a-helical in nature. Broadening of the spectrum between 215–225 nm may arise from contributions of b-sheet and a-helix to the spectra. As the CD spectra of protofibrils of other amyloid-forming proteins is characterised by b-sheet absorption at 215–218 nm [61,71,72], it is likely that the MstnPP soluble aggregates represent an oligomeric intermediate state prior to protofibril formation rather than protofibrils. Incubation of the aggregates overnight at pH 5.3 and 60uC produces a CD spectrum dominated by b-sheet where the a-helix minimum at 208 nm is completely replaced by a minimum at 218 nm. TEM images of the same sample (Fig. 4) reveals there are no fibrils at this stage but the aggregates are more compact and have begun to aggregate into large arrays, which may represent the start of b-sheet stacking as amyloid protofibrils. The ellipticity for the spectrum is comparatively low, similar to that observed in the fibrillogenesis of Ab [65]. This reduction of signal is most likely due to differential absorption flattening, a phenomenon often seen in the CD spectra of samples containing suspensions of solid-phase material [61] and causing both a decrease in intensity and red-shift of all minima. After one week of incubation the spectrum is similar except for flattening of the 190 nm peak. At this stage, fibrils are present as observed by TEM analysis of an aliquot of the same sample (Fig. 4). The further flattening of the spectrum over time is likely to be due to both an increase in b-sheet structure as well as differential absorption flattening [61,69]. CD shows that the soluble aggregates differ dramatically in secondary structure compared to native MstnPP and that the aggregates may represent an a-helical-containing intermediate in the transition to protofibrils and fibrils rich in b- sheet structure. MstnPP Protofibrils and Fibrils Are Resistant to Proteolytic Digest by Trypsin Resistance to proteolytic digestion is another defining charac- teristic for the presence of amyloid fibrils [73,74]. Comparative trypsin digestions were performed for the MstnPP fibril-containing sample, soluble aggregates and native dimer (Fig. 6). Digestion of the MstnPP dimer and aggregates was carried out with a MstnPP to trypsin ratio of 100:1 (w/w) at 4uC, room temperature (approximately 22uC) and 37uC with samples taken after 0.5, 1, 2, 3, 4 and 18 hours. The precursor dimer and aggregates have comparable trypsin susceptibility at 37uC and are fully digested after an overnight incubation (18 hours, Fig. 6A and B respectively). The susceptibility of the soluble aggregates to trypsin digest suggests an open, flexible structure, consistent with CD results and supporting the conclusion that this species represents a prefibrillar intermediate rather than protofibrils. Prior to digestion the fibril-containing sample is not able to enter the top of the 4% PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9170