FTIR Analysis of Protein Secondary Structure

FTIR was performed with a Jasco FT/IR6200 spectrometer (JASCO, Tokyo, Japan) equipped with a MIRacle attenuated total reflection (ATR) Ge crystal cell in reflection mode. For each sample, 32 scans of 4 cm⁻¹ resolution were coadded and Fourier transformed using a Blackman- Harris apodization function. The amide I region (1585 to 1720 cm⁻¹ ) was deconvoluted and peak fitted using Opus 5.0 software (Bruker, Billerica, MA) to characterize the secondary structure content (side chains, β-sheet, random coil, a-helix and β-turns) as previously described.^{41 –43 (link)} The relative contributions of the secondary structure to the C=O stretch were quantified. Briefly, the FTIR spectra obtained from the instrument were cut and baselined between 1750 and 1150 cm⁻¹, Fourier self-deconvoluted between 1720 and 1585 cm⁻¹ using a bandwidth of 27.5 cm⁻¹, noise reduction of 0.3 and a Lorentzian line shape, then baselined again between 1710 and 1585 cm⁻¹ and the peaks corresponding to a local minimum in the second derivative were curve fitted using a Levenberg-Marquardt algorithm and local least-square analysis. The relative peak areas were assigned to different secondary structure contributions based on the peak locations and reported as a percentage of the total peak area.