Continous gradient temperature Raman Spectroscopy identifies flexible sites in proline and alanine peptides

Continuous gradient temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry (DSC) to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements that occur near phase transitions. Herein we apply GTRS and DSC to the solid dipeptides Ala-Pro, Pro-Ala, and the mixture Ala-Pro/Pro-Ala 2:1. A simple change in residue order resulted in dramatic changes in thermal stability and properties. Characteristic Pro vibrations were observed at ∼75 °C higher temperature in Pro-Ala than Ala-Pro. The appearance/disappearance of characteristic vibrational modes with increasing temperature showed that a double peak in the Ala-Pro major phase transition (174–184 °C) was due to a gauche to anti 165° rotation of H3CC*NH3 about C*. CH2 rocking and wagging frequencies present in Pro-Ala were not observed in Ala-Pro. For Ala-Pro, the Ala +NH3, and Pro COO− sites were flexible whereas the Pro ring moiety was not; since the OCN (C)2 amide bond is planar the CNC moiety keeps the Pro ring rigid. For Pro-Ala, CH2 sites in the Pro ring were flexible and the OCNH amide bond is perpendicular to the Pro ring. Since the mass of the Pro ring is significantly larger than the mass of the flexible Ala +NH3 moiety, Pro-Ala absorbs more thermal energy, corresponding to a higher phase transition temperature (240–260 °C). Ala-Pro, Pro-Ala, and Ala-Pro/Pro-Ala 2:1 exhibited α-helix, β-sheet, α-helix secondary structure conformations, respectively.