(238e) The Relationship between Flexible Peptide Conformation (from unfolded PPII to fully extended beta-sheet) and Crystallisation Conditions

Intrinsically disordered proteins (IDP) make up more than 30% of the human genome, and these unstructured regions are often key to the function.¹ pPII conformation, as a significant contributor in the unfolded peptide structures, has been observed to be preferred in proline-, alanine- and glycine-rich regions in peptides and proteins. The presence of water has been proven can stabilize the pPII conformation in unfolded regions of proteins. However, previous research has not given us a clear and consistent result about how water interacts with peptides to stabilize the pPII conformation and how the crystallisation conditions (under different pH, solvents, and salts) effect the flexible conformation and the final stable crystalline form.²

Here, we show for the first time a glycine homopeptide (gly-gly-gly) adopting the pPII conformation in its crystalline dihydrate structure.³ The single crystal X-ray structure with molecular dynamic simulation suggests that even though the central glycine residue can contribute to the pPII conformation, the carboxylate-water and water-water interactions play a more important role in stablizing the pPII conformation. High temperature (above 303.15K), low water activity (high ethanol ratio), and high salts (LiCl, NaCl, KCl, MgCl₂, K₂SO₄, Li₂SO₄, Na₂SO₄ and K₂SO₄) concentration were found to have a negative effect on the PPII conformation in the crystalline form. The critical ethanol ratio and salt concentration under different temperatures have been explored here to show the suitable conditions for the PPII conformation and triglycine dihydrate. The pH values under different conditions have been measured to explore a general mechanism of the formation of pPII under the above conditions. MD simulations under the above variable conditions have been done to create the Ramachandran plot which shows the distribution of the variable conformations in the solution state. The simulation results were also verified by the ReactIR spectrum which monitored the peptide conformation change during the phase transformation and crystallisation process. From the results, the conformation in both MD simulation and IR spectrum in the liquid state corresponds to that in the final solid state. Figure 1 shows the decreasing probability of the PPII conformation with the increasing ratio of ethanol in water from the MD simulation, which means the conformation in the liquid states determined the final conformation in the crystalline form.

This research gives a comprehensive understanding of the transition between protein unfolded states (PPII) and folded states (extended beta-sheet) in solid and liquid states, and provides insight into the effect of the environment on the flexible conformations and structural stability of peptides during the macromolecular crystallisation.

1. Wei, L.; Zhao, Y.; Hu, X.; Tang, L., Redox-Responsive Polycondensate Neoepitope for Enhanced Personalized Cancer Vaccine. ACS Cent Sci 2020, 6 (3), 404-412.

2. Brian, A.; Shuting, Z.; Reinhard, S.-S.; Brigita, U., Glycine in Water Favors the Polyproline II State. Biomolecules 2020, 10, 1121-1141.

3. 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.