810 spectropolarimeter

Homology modelling of the periplasmic/outer membrane component of Bucl8 was
performed using the software MODELLER [32 (link)] and the structure of VceC from
V. cholerae as a template (PDB code 1yc9).
For the collagen-like (CL) region of Bucl8, homology modelling was performed
with MODELLER [32 (link)] using
the high-resolution structure of a collagen-like peptide (PDB code 1k6f) [33 (link)] as a template. The Ct
random coil region was generated using the Molefacture plugin of VMD [34 (link)]. Electrostatic
potential surface was computed using the software Chimera [35 (link)].
Circular dichroism spectroscopy (CD) of rBucl8-derived polypeptides was performed
as previously described [30 (link)]. Briefly, protein samples were dialyzed against 1x Dulbecco’s
phosphate buffered saline, pH 7.4. CD spectra were taken with a Jasco 810
spectropolarimeter, in a thermostatically controlled cuvette, with a path length
of 0.5 cm. Data were acquired at 10 nm per minute. Wavelength scans were
performed from 240 nm to 190 nm at either 25°C or 50°C for unfolded triple helix
in rBucl8-CL-Ct construct.

Peptide samples were prepared by diluting stock solutions immediately before measurements to a concentration of 100 μM in 2,2,2-trifluoroethanol (TFE) 5% (soluble samples) or in PBS (aggregated samples). CD spectra were recorded at the indicated times of incubation, scanning from 260 to 190 nm on a Jasco 810 spectropolarimeter equilibrated at 25 °C. Each spectrum shown is the accumulation of 10 scans.

Samples of recombinant PrP at concentrations of 25 μM prepared in 50 mM sodium acetate were used for CD spectroscopic analysis. All samples were centrifuged at 13,000 ×g at 4°C for 30 minutes prior to analysis. CD spectra were recorded in a 0.5 mm path-length quartz cuvette at 20°C, under constant nitrogen flushing using a JASCO 810 spectropolarimeter. At least 10 spectra were accumulated and the values were expressed as molar ellipticity (θ). Secondary structure content was determined from deconvoluted CD spectroscopic data using the CDNN programme [39 (link), 40 (link)].

Circular dichroism (CD) spectra of synthetic hFWE fragments were recorded by using a JASCO 810 spectropolarimeter (Tokyo, Japan). Peptides were dissolved in 25 mM phosphate buffer, 37.5 mM NaCl, pH 7.4, and the concentration of the stock solutions were determined by quantitative RP-HPLC analysis (23 (link)). Peptide solutions were diluted to 50 μM, except the concentration of Ac-FWE(51–57)-NH₂ was 200 μM. Because of its hydrophobicity, Ac-hFWE4(121–142)-NH₂ was dissolved in 33% ACN in water. CD spectra of Ac-hFWE(51–57)-NH₂, Ac-FWE(84–95)-NH₂, hFWE4(121–142)-NH₂ and Ac-hFWE4(145–172) were also measured in the presence of 15%, 30% and 45% TFE in same buffer (v/v). The samples were loaded onto a 0.05-cm path length cell, and the temperature of the solutions was kept at 25 ^°C by using a Jasco PFD-425 Peltier thermostated cell holder. The sensitivity was set to normal, and samples were scanned from 185 to 260 nm wavelengths at a scan speed of 100 nm/min with 0.1 nm data pitch. CD spectra were obtained by averaging 20 scans and subtracting the solvent baseline. To calculate secondary structure composition, each CD spectra was deconvoluted by the CDSSTR algorithm with the basis set 4 as reference using the DichroWeb CD analysis deconvolution server (24 (link)).