Characterization of Secondary Structures of Model Polypeptides in Solutions with Hyper-Raman Spectroscopy

Characterization of the secondary structures of two model polypeptides, poly-l-lysine and poly-l-glutamic acid in aqueous solutions has been demonstrated by hyper-Raman (HR) spectroscopy for the first time. Complementary to infrared (IR) and visible Raman spectroscopy, HR spectroscopy gives the amide I, II, and III bands originating from the polypeptide backbones and the CCH₃ symmetric bending mode, enabling us to distinguish different conformations. The α-helix gives the broad and weak amide III band, while the β-sheet and the random coil show similar spectral patterns with different relative intensities between the amide I and II bands. HR spectra from aqueous solutions of the α-helix and the random coil of poly-l-ornithine also possess these spectral features. The HR spectra are analogous to UV resonance Raman (UVRR) spectra, indicating the signal enhancement due to the electronic resonance effect via the π-π* transition. In contrast, the vibrational frequencies of the amide I band in the HR spectra are much higher than those in the IR, visible Raman, and UVRR spectra, suggesting the non-coincidence between HR, IR, and Raman bands. Our finding suggests that HR spectroscopy is promising to provide complementary information on the secondary structures of polypeptides in aqueous solutions as a spectral approach differing from existing vibrational spectroscopic methods.