Peptide CD Data Collection and Secondary Structure Prediction

Secondary structure estimation was accomplished using a Jasco J-810 circular dichroism (CD) spectrophotometer. Solutions of 40μM peptide in 100mM Tris-HCL buffer with varying volumes of 2,2,2-trifluoroethanol (TFE) were prepared for CD analysis. A minimum of 8 scans over wavenumber 190–260nm with a scan rate of 0.5 nm/min were collected on a calibrated spectrophotometer and averaged. The background was subtracted, and the spectra smoothed using the Savitzky-Golay algorithm. The resulting CD spectra were deconvoluted using the BeStSel web server for accurate prediction of protein secondary structure and folding ⁷⁰.

Predicted secondary structure contents for helical (α, 3₁₀ and π-helix), beta (β-bridge, bonded turn), and irregular (bend and loop) features were determined using the Chou-Fasman algorithm. The Chou-Fasman algorithm was applied for each bifunctional peptide after uploading their CD spectra and amino acid sequences using the online server available through the CD Analysis and Plotting Tool (CAPITO) ⁷¹.