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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Environmental Microbial & Food Safety Laboratory » Research » Publications at this Location » Publication #316962

Research Project: DEVELOPMENT OF SENSING AND INSTRUMENTATION TECHNOLOGIES FOR FOOD SAFETY AND SANITATION INSPECTION IN FRESH FRUIT AND VEGETABLE PROCESSING

Location: Environmental Microbial & Food Safety Laboratory

Title: Parallelism between gradient temperature raman spectroscopy and differential scanning calorimetry results

Author
item Nguyen, Julie
item Schmidt, Walter
item Broadhurst, Leigh - University Of Maryland
item Kim, Moon
item Qin, Jianwei - Tony

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 5/5/2015
Publication Date: 5/20/2015
Citation: Nguyen, J.K., Schmidt, W.F., Broadhurst, L., Kim, M.S., Qin, J. 2015. Parallelism between gradient temperature raman spectroscopy and differential scanning calorimetry results. BARC Poster Day. BARC Poster Day on May 20, 2015..

Interpretive Summary: Although flexibility in peptide and protein structures is critically important, identifying flexibility at multiple molecular sites simultaneously is elusive. In particular, the structural changes involved in the thermal adaptation of proteins (i.e. response to heating) are manifold and complex. Raman spectroscopy has previously been used to elucidate molecular structure at constant temperatures (i.e. steady state). We have developed a novel temperature dependent Raman spectroscopy (TDR) technique that measures Raman spectroscopic responses of sample materials in precise temperature gradients. The technique provides a rapid means to identify molecular rearrangements that occur just prior to thermal phase transitions. In this investigation, we applied the TDR technique to the dipeptide structural analogs Ala-Pro and Pro-Ala, and to a mixture of the two; proline is of particular interest because it is an atypical amino acid that is prevalent in thermophilic proteins. All three exhibited different phase-transition temperature profiles. The results showed markedly different Raman responses, unraveling flexible molecular sites under thermal stress. 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. The technique and information provided in this study will benefit biochemists and others researching the dynamics of protein unfolding.

Technical Abstract: Temperature dependent Raman spectroscopy (TDR) 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 TDR and DSC to the dipeptides Ala-Pro and Pro-Ala, and to 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. 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 and 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, and ''helix secondary structure conformations, respectively.