Figure 1. In silico predictions of propensity for b-sheet aggregation (Tango and PASTA) and amyloid formation (Waltz). A. MstnPP sequence 21–375 showing regions of elevated propensity at two levels; red regions have higher propensity than those in blue. Regions where Waltz, Tango and PASTA predictions overlap are underlined. The RSRR furin proteolysis sequence that separates the propeptide from the growth factor domain is shown in bold. B. Mapping the 348–364 sequence (blue) onto the myostatin growth factor crystal structure. The region has been shown on one monomer only. doi:10.1371/journal.pone.0009170.g001 Western blotting identifies both peaks as myostatin (Fig. S2B) but the two species interact differently with the heparin column suggesting a difference in folding. Further purification of both peaks was achieved using gel filtration (Fig. 2C). The aggregates are too large to move from the well into an SDS-PAGE gel (above 250 kDa, Fig. 2A), elute in the void volume during S200 gel filtration (PA) (Fig. 2C) and remain soluble even after ultracen- trifugation at 214,0006g (data not shown). Since the MstnPP monomer is 43 kDa the soluble aggregates are most likely to be an oligomer of at least 5 monomeric units. MstnPP Soluble Aggregates Have Characteristics of Amyloid-Like Protofibrils Transmission electron microscopy (TEM, Fig. 3A and B) revealed that MstnPP soluble aggregates exhibit a morphology and size similar to that documented for amyloid protofibrils from a number of other proteins such as lysozyme [59,60] and HypF-N [61] as well as the insulin protofibrils generated as a positive control (Fig. 3C) using a well-established protocol [39,62,63]. A range of structures, including spheres and oligomers of associated spheres (diameter 15.661.6 nm SD), can be seen, and examples of increased elongation of the aggregates are apparent (Fig. 3B). A defining characteristic of amyloid fibrils and protofibrils is their ability to bind the fluorescent dye thioflavin T (ThT) [63–65]. When bound to fibrils, ThT can be excited at 450 nm to fluoresce at 485 nm [66]. One limitation of this method is the lack of a strict quantitative relationship [64] with amyloid fibrils often producing a stronger emission than protofibrils by comparison [65]. ThT Myostatin Amyloid Formation binding assays were carried out on the MstnPP aggregates and compared to a solution containing a mixture of insulin protofibrils and fibrils as a positive control. The MstnPP soluble aggregates bound ThT with intensity comparable to insulin fibrils whereas the correctly folded MstnPP dimer did not (Fig. 3D). These results suggest that a population of MstnPP can aggregate spontaneously to form amyloid-like protofibrils. Formation of MstnPP Amyloid Fibrils at Acidic pH and Elevated Temperature The presence of MstnPP amyloid-like protofibrils suggests that amyloid fibril formation is also possible. MstnPP aggregates were concentrated and resuspended in dilute HCl solutions (pH range 1.6 to 6.3) and incubated at either 37u or 60uC, conditions including those under which insulin forms amyloid fibrils [62,63]. Solutions were monitored by ThT binding and TEM (Fig. 4). After one week at 60uC, ThT fluorescence had increased significantly in the pH 5.3 solution (Fig. 4A), which contained a mixture of prefibrillar aggregates and amyloid fibrils observed by TEM (Fig. 4B–4F) in both the presence and absence of 0.1% sodium azide. The fluorescence assays were also performed by diluting protein solutions into a pH 7.5 buffer since ThT binding may be affected by changes in pH [67]. In these assays, fluorescence values for the soluble aggregates were largely unchanged but an increase in intensity for the pH 5.3 samples was observed (data not shown), consistent with the lower pH results. Myostatin fibril formation occurs via a number of stages. For a 3 mg/mL solution, overnight incubation at 60uC in pH 5.3 produces oligomers that are compact and have a ‘bead-on-a- string’ morphology (Fig. 4B) as observed for Ab and lysozyme fibrils [60,65]. By three days, extensive arrays of aggregation were present; these are often three-dimensional, appearing as areas of high electron density under the TEM (Fig. 4Ci). Increased elongation and in some places lateral association and longitudinal fusion of oligomers can be observed (Fig. 4Cii and 4Ciii). This granular to smooth transition has been shown during Ab fibril formation [65]. Smaller pore-like oligomers, previously document- ed on the amyloid formation pathway of a number of proteins [40,43], are also apparent (Fig. 4Civ and 4Cv). By one week, fibrils that show the characteristic morphology of amyloid had appeared, with a diameter of 15.460.7 nm (SD) and a straight, unbranching structure (Fig. 4D). The fibrils are extremely long, in excess of 5 mm. At two weeks, higher order amyloid structures can be observed, such as two (Fig. 4E) or more (Fig. 4F) fibrils twisting around each other. Secondary Structure Analysis of MstnPP Fibril Formation by Circular Dichroism Spectroscopy Amyloid formation is accompanied by the adoption of a b-sheet rich secondary structure regardless of the structure of the native protein [68]. Circular dichroism (CD) spectroscopy was used to study the structural changes that occur during amyloid formation (Fig. 5). Correctly refolded MstnPP has a CD spectrum indicative of a mixture of a-helix and b-sheet, in agreement with the crystal structure of the mature myostatin growth factor [11] and the mature regions of other TGF-b family members [57]. There are two minima, the stronger at 218 nm and a second at 208 nm which are characteristic of b-sheet and a-helical structures respectively [69]. The absolute negative value at 200 nm implies a degree of intrinsic disorder exists in the structure [65,70], which may be functionally significant due to the different roles suggested for the propeptide region of the myostatin precursor, requiring flexibility in the N-terminal domain. To our knowledge, this is the PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9170