Enhanced characterization of protein secondary structure transitions using Raman and SERS measurements combined with...

Raman spectroscopy is a valuable tool for characterizing the secondary structure of proteins, with surface-enhanced Raman spectroscopy (SERS) further amplifying the signals. However, these techniques often face challenges due to the broadening of amide bands associated with complex protein motifs and the suppression of amide bands from interactions between proteins and SERS substrates. Herein, we employed Raman and SERS measurements in conjunction with 2D correlation spectroscopy (2D-CoS) and principal component analysis (PCA) to investigate the structural transitions of alpha-helical peptides, beta-sheet peptides, and helical bundles of SNARE protein from their native folded states to unfolded states. Our findings reveal an inverse relationship between amide I band shifts and hydrogen bonding strength in the protein backbone. Notably, the 2D correlation spectroscopy implies a positive correlation of the amide I band (1650-1680 cm^-1), the amide III band (1230-1320 cm^-1), with the methyl deformation bands (1440-1460 cm^-1) along with the changes of protein secondary structures. This suggests that the strong and reproducible methyl deformation bands at 1440-1460 cm^-1 may serve as reliable indicators of protein secondary structure, especially when the amide I bands are suppressed or difficult to resolve from SERS measurements. Furthermore, the enhanced analysis of the vibrational modes of native folded, unfolded, and refolded SNARE proteins using PCA and 2D correlation analysis can differentiate its reversible unfolding and irreversible unfolding pathways. Our vibrational analysis approaches show great promise for fully leveraging Raman and SERS techniques to monitor native protein structural transitions during dynamic folding and unfolding processes at physiological conditions.