810 circular dichroism spectropolarimeter

Experimental characterization of the average secondary structure of the various peptides was conducted via circular dichroism spectroscopy on a Jasco 810 circular dichroism spectropolarimeter (Jasco Inc, Easton, MD, USA). CD spectra were recorded using a quartz cell with 0.1 cm optical path length. Samples for full wavelength scans incubated at various temperatures were cooled for three minutes at 0 °C prior to the start of the experiment. For each wavelength scan, the scanning rate was 50 nm/min, with a response time of 4 s. Wavelengths from 195 nm to 250 nm were recorded at increments of 0.5 nm. (MRE) values at 222 nm ([Θ]_MRE,222) were used to calculate fraction of α-helicity was calculated by using a previously established method that is based on idealized long helices.[34 (link)]

Circular dichroic spectroscopy (Jasco 810 circular dichroism spectropolarimeter, Jasco Inc., Easton, MD) was conducted for the characterization of the secondary structure of the CLP domain. CLP and ELP−CLP conjugates were dissolved at a concentration of 100 μM in PBS (10 mM, pH 7.4, 137 mM NaCl and 2.7 mM KCl) and incubated at 4 °C overnight before measurement. The CD spectra were recorded using quartz cells with a 0.2 cm optical path length. Full wavelength scans were collected to study the conformation of the peptide domain at 4 °C. The scanning rate was 50 nm/min, with a response time of 4 s. The wavelength scans were obtained from 200 to 250 nm and were recorded every 1 nm. To measure the melting temperature of the CLP domain, variable temperature experiments were conducted at the maximum wavelength in each ELP−CLP conjugate (e.g., 225 nm) with a 10 °C/h heating rate from 4 to 80 °C