Peptide Structural Changes

To determine the structure of CRAC^336WT after binding to Chol and other sterols, circular dichroism (CD) spectra were recorded using a Jasco J-815 CD spectrometer (Jasco Inc., Easton, MD). Spectral scans were performed from 240 to 190 nm, with a scanning speed of 20 nm/min and a bandwidth of 1.0 nm, in 10 mM phosphate buffer using a peptide concentration of 0.25 mg/mL. A 0.01 cm path-length quartz cuvette was used for the measurements. To ensure that the spectra represent the structure of only bound peptide, unbound peptide was removed using centrifugal filters (Amicon 30000 MWCO, EMD Millipore) after 30 min.^33,34 CD spectra were processed in OriginPro2015. The secondary structure was determined with DICHROWEB using CONTIN/LL and either the SP175 reference set (for solutions containing only peptide) or the SMP180 reference set (for solutions containing peptides and liposomes).^35-40