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2.7.1 Circular dichroism spectroscopy

CD spectra in the far-UV region (180-260 nm) were obtained on a Chirascan CD spectrometer (Applied Biophysics) using a 0.1 mm path cell and protein concentrations of 1 mg/mL, or a 1 mm path cell and protein concentrations of 0.1 mg/mL, at 4 °C. For all samples, 20 runs were performed with 1 nm readings taken every 2.5 seconds, followed by smoothing and baseline subtraction. Deconvolution of CD spectra was performed using CDDN software (Applied Biophysics) (Appendix 1, Table 12.1 and Appendix 4, Table 12.2a and b).

2.7.2 CD thermal denaturation

For CD thermal denaturation, 1 nm/2.5 second readings were taken at every 5 °C (precursor protein and soluble aggregates) or 10 °C (latent complex) increase in temperature from 5 – 90 °C with a 30 second equilibration time at each temperature and a tolerance level of 0.2 °C.

2.7.3 Thermal shift assays

Purified WT or C313Y MstnPP in 50 mM Tris-HCl, 150 mM NaCl, pH 8.5 was concentrated to 1.0 mg/mL. Sypro Orange (Sigma) was diluted 100x according to manufacturer’s instructions in milliQ H2O and 2 μL was added to 18 μL of protein

solution, to give a final dilution factor for the dye of 1000 x. A negative control containing buffer and dye only was also made. The RotorGene 6000 thermocycler (Corbett) was used for analysis with excitation at 470 nm and fluorescence emission measured at 555 nm over increasing temperature from 30 to 95 °C in 1 °C increments;

each run lasted approximately 19 minutes. Differentiation of the data was performed by the Rotor Gene 6000 Series software.

2.7.4 X-ray crystallography

Purifed MstnPP dimer was concentrated to 5 mg/mL, centrifuged at 17,000 x g for 10 minutes at 4 °C and filtered (0.22 μm) for crystallisation trials. 1 μL of protein was mixed with 1 μL of mother liquor in sitting drops using 96-well Crystalquick plates (Greiner Bio-One) containing 100 μL well volumes of Crystal Screen and Crystal Screen 2 (Hampton Research) at 22 °C. Purified lyophilized myostatin growth factor (MstnGF) was resuspended in 10 mM acetic acid to a concentration to 3 mg/mL, centrifuged at 17,000 x g for 10 minutes at 4 °C and filtered (0.22 μm). 200 nL of protein was mixed with 200 nL of mother liquor by the Mosquito crystallisation robot (TTP Labtech) at Otago University in a hanging drop format. Plates contained 100 μL wells of Crystal Screen and Crystal Screen 2 (Hampton Research) and Structure Screen I and II (Molecular Dimensions) in a 96-well format. For both proteins, optimisation of conditions was done by the hanging-drop, vapour-diffusion method using a range of protein to mother liquor (v/v) ratios; for the growth factor a ratio of 2:1 was used. Crystals were screened for diffraction in-house using an RAXIS IV++ image plate coupled to a MicroMax-007 X-ray generator (Rigaku) with Osmic Blue optics. Diffraction was monitored using Crystal Clear software (Rigaku). Crystals were imaged and photographed using a Zeiss Axiophot compound light microscope fitted with a Leica DFC320 digital camera, and Leica Application Suite V3.3.0 software.

2.7.5 Protease resistance analysis

MstnPP samples were tested for protease resistance using a myostatin to trypsin (bovine pancreas, Sigma) ratio of 100:1 (MstnPP WT and C313Y dimer and soluble aggregates), or 20:1 (MstnPP protofibrils and fibrils) (w/w). 100:1 solutions were incubated at 4 °C, room temperature or 37 °C. Samples were taken at 0.5, 1, 2, 3, 4 and 18 hours (overnight), immediately denatured to end the reaction and analysed by reducing and non-reducing SDS-PAGE. For additional protease resistance analysis of MstnPP amyloid protofibrils and fibrils, the myostatin to trypsin ratio was 20:1 (w/w) with incubation at 37 °C for 4 and 18 hours and analysis by SDS-PAGE and TEM.

2.7.6 Negative-stained transmission electron microscopy

Dimer and aggregate samples direct from purification (heparin affinity or gel filtration) were buffer-exchanged into water. Amyloid solutions were used directly in dilute HCl. 200-mesh carbon-coated Formvar grids were placed on 30 μL protein solution (0.5 mg/mL) droplets for 45 seconds. Excess sample was drawn off using filter paper and the grids placed on an equal volume of 2% uranyl acetate for 45 seconds. Excess stain was drawn off as above and the grids were air-dried briefly before viewing with a Philips CM10 transmission electron microscope. Fibril width measurement and statistical analysis was performed using iTEM software (Olympus) with a sample size of 10 fibrils from the same grid. Where a single fibril was concerned, at least 5 measurements along the same fibril were taken. For twist distance measurement and analysis, a sample size of at least 5 twists from the same same fibril was used.

2.7.7 Thioflavin T binding assays

A solution of Thioflavin T (Sigma) was prepared at 400 μM in water (Bourhim, Kruzel et al. 2007) then diluted directly into the protein solution giving a final concentration of 20 μM per assay, as done previously for insulin (Groenning, Norrman et al. 2007)). A final protein concentration of 1 mg/mL was used for all samples. Experiments at pH 7.5 were performed after diluting both protein and ThT into 50 mM Tris-HCl pH 7.5, 100 mM NaCl. Fluorescence assays were conducted with the Perkin Elmer LS50B Luminescence Spectrometer with excitation at 450 nm and the emission spectrum measured from 460-530 nm. Error bars are standard errors for four independent measurements. The Student’s t-test was performed using GraphPad Prism (GraphPad Software, Inc).

2.7.8 One dimensional NMR

1D NMR experiments were performed on a Bruker Avance 700 MHz spectrometer equipped with a cryoprobe, four rf-channels and gradient pulse capabilities. All spectra were acquired at 25 oC on 300 μL samples containing 5% D2O in Shigemi

NMR tubes. The myostatin growth factor concentration was 0.2 mM in 50 mM Na acetate, pH 4, 100 mM NDSB 201. 1H spectra were recorded with 32 transients, and 160000 points with a spectral width of 8389.2 Hz. Fourier transformation and final

processing was accomplished using TopSpin 2.1 (Bruker BioSpin GmbH, Rheinstetten, Germany).