Gradient temperature Raman spectroscopy identifies flexible sites in proline and alanine peptides

Authors: Schmidt, Walter; Kim, Moon; Nguyen, Julie; QIN, JIANWEI - University Of Maryland; Chao, Kuanglin - Kevin Chao; LEE, HOYOUNG - US Department Of Agriculture (USDA); Shelton, Daniel

Submitted to: Vibrational Spectroscopy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/18/2015
Publication Date: N/A
Citation: N/A

Interpretive Summary: Although flexibility in peptide and protein structures is critically important, identifying flexibility at multiple sites simultaneously is elusive. In particular, structural changes involved in the thermal adaptation of proteins are considered manifold and complex. Every day, billions of people consume animal or plant proteins subjected to thermal stress (i.e. cooking, canning, processing); yet quantifying the processes involved in the thermal transformation of proteins remains as much a culinary art as a science. Proline (Pro) is an atypical amino acid with the N atom part of a five-membered ring. Proline contains a +NH2 zwitterion site instead of +NH3, and the angular range in peptide bond formation is highly restricted; when inserted in peptides it can cause “kinks” in their structures. Proteins that are very heat stable have relatively higher concentrations of Pro than those that are not. Overall, proteins are universally stabilized by Pro residues at the N-terminal of an '-helix, regardless of their degree of thermostability. Our research group has developed the technique of continuous temperature dependent Raman spectroscopy (TDR), which applies the precise temperature gradients utilized in differential scanning calorimetry (DSC) to Raman spectroscopy. TDR provides a very rapid and straightforward technique to identify theoretically proposed molecular rearrangements that occur just prior to phase transitions. In this contribution we applied the TDR technique to the dipeptide structural analogs Ala-Pro and Pro-Ala, and the mixture Ala-Pro/Pro-Ala 2:1; and correlate the results with DSC. DSC results showed Ala-Pro has a relatively sharp and reversible phase transition (174–184oC) with two distinct regions. A broader transition follows at 206-256oC. Pro-Ala has a single sharp phase transition temperature region between 240-260oC. The DSC data for Ala-Pro/Pro-Ala 2:1 yields a curve that is intermediate between the two end members, indicating that Pro-Ala and Ala-Pro interacted as they were heated. The TDR spectra of Ala-Pro and Pro-Ala also differed markedly. CH3 asym bending and CH2 rocking and wagging frequencies present in Pro-Ala are not observed in Ala-Pro. Many characteristic Pro vibrations were observed at ~75oC higher temperature in Pro-Ala than those same vibrations in Ala-Pro. The appearance/disappearance of characteristic vibrational modes with increasing temperature showed that the double peak in the Ala-Pro phase transition (174–184oC) was due to a 165 degree rotation of the NH3 group. For both dipeptides, the amino acid with the free +NH site was routinely more flexible. Flexible sites absorb thermal energy faster and have more available vibrational modes. For Ala-Pro, the Ala +NH3, and Pro COO- sites were flexible whereas the Pro ring moiety was not; since the O=C-N(-C)2 amide bond is planar the C-N-C moiety in the ring keeps the remainder of the Pro ring rigid. Thermal energy is also involved in intermolecular bonding. Vibrations perturbing the flexible +NH3 and Pro COO- in Ala-Pro disrupts the crystal structure of the peptide, which further acts to lower phase transition (melting/decomposition) temperatures. In contrast, for Pro-Ala, CH2 sites in the Pro ring were flexible, but flexibility was not achieved until over 230oC, long after Ala-Pro has melted/decomposed. Since the mass of the (eventually) flexible Pro ring is significantly larger than the mass of the flexible Ala +NH3 moiety, Pro-Ala can absorb more thermal energy, which corresponds to its higher phase transition temperature. Thus a simple change in residue order of the dipeptide results in dramatic changes in thermal stability and properties. We are the first research group to provide a molecular rationale for how thermophilic proteins can become more flexible at the relatively higher temperatures at which they function, and for the stabilizin

Technical Abstract: Continuous thermo dynamic Raman spectroscopy (TDRS) applies the temperature gradients utilized in differential scanning calorimetry (DSC) to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements that occur just prior to phase transitions. Herein we apply TDRS and DSC to the 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 ~75oC 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 (174–184oC) was due to a 165 degree rotation of H3C-C*-NH3 about C*. CH3 asym. bending and 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 O=C-N (-C)2 amide bond is planar the C-N-C moiety keeps the Pro ring rigid. For Pro-Ala, CH2 sites in the Pro ring were flexible; the O=C-NH amide bond is perpendicular to the Pro ring thus +NH3 frequencies 650-850 cm-1 were not observed. 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 (240-260oC). Ala-Pro, Pro-Ala, and Ala-Pro/Pro-Ala 2:1 exhibited ''helix, '-sheet, ''helix secondary structure conformations, respectively.