(488a) Exploring the Peptide Conformation Change during the Dehydration Process: A Case Study of Triglycine Dihydrate

The crystal structure of biomolecules provides insight into their internal structures but represents only one stable conformation. Although the final crystalline form can offer insights into interactions between proteins and other molecules like water and ligands, it is still uncertain how these interactions may alter during the crystallization or storage process. Moreover, most proteins have water molecules inside their crystal structure, and the water content change during storage can impact the stability of the conformation and crystal structure.¹

In our previous study, triglycine was observed to adopt a fully extended beta-sheet conformation in the anhydrate form but an unfolded (pPII) conformation in the hydrate form.² The pPII conformation is a novel conformation observed in unfolded peptides and proteins, while the fully extended beta-sheet conformation is characteristic of folded peptides and proteins. The unique conformations of triglycine make its dihydrate form an excellent model for investigating the dehydration process and exploring whether the conformation can revert to the extended beta-sheet conformation after the water loss. This research aims to explore the dehydration process of peptide hydrates and study the effect of the drying process on the final conformation and crystalline form.

The raw sample of triglycine dihydrate was subjected to TGA analysis from 298.15 K to 850 K at a heating rate of 10 K/min. A constant nitrogen flow of 60 mL/min was used to prevent thermal oxidation processes. It was found that water was lost before 60°C (Figure 1). Based on these results, the dehydration process was designed to expose the sample to 60°C for 24 hours. Subsequent TGA analysis showed that no water remained in the dehydrated sample (Figure 1), with PXRD analysis providing evidence for the anhydrate form. Variable temperature PXRD was used to track the transformation of the crystal structure from triglycine dihydrate at 40°C to 100°C with the temperature rising rate at 2K/min, revealing a clear trend toward the anhydrate form (Figure 2). Additionally, FTIR was utilized to measure conformational changes before and after dehydration, with the spectrum of the dehydrated sample displaying some new peaks consistent with the anhydrate form, indicating a conformational transition from the unfolded PPII to beta-sheet conformation (Figure 3 and 4). This finding offers a foundation for investigating the impact of water content on peptide crystal stability by demonstrating conformational changes during the dehydration process.

Overall, this study shows the trend that the unfolded PPII conformation can convert back to the fully extended beta-sheet conformation upon water loss, highlighting the crucial role of water in stabilizing the PPII conformation not only in the liquid state but also in the solid state.

1. Xuan, T.; Bogdanova, E.; Fureby, A.; Fransson, J.; Terry, A.; Kocherbitov, V., Hydration-Induced Structural Changes in the Solid State of Protein: A SAXS/WAXS Study on Lysozyme. Mol Pharm. 2020, 8; 17(9):3246-3258

2.Guo, M.; Rosbottom, I.; Zhou, L.; Yong, C. W.; Zhou, L.; Yin, Q.; Todorov, I. T.; Errington, E.; Heng, J. Y. Y., Triglycine (GGG) Adopts a Polyproline II (pPII) Conformation in Its Hydrated Crystal Form: Revealing the Role of Water in Peptide Crystallization. J Phys Chem Lett 2021, 8416-8422.