Publicação
Probing the Stability of Biological Molecules in Deep Eutectic Solvents
| Resumo: | Deep eutectic solvents (DESs) have gained prominence as promising alternative solvent media in biocatalysis and biomolecular (cryo)preservation for fragile biomolecules, such as DNA, RNA, cells (including stem cells), and even organs intended for transplantation. The stabilization of proteins by osmolytes, common components of natural DESs (NADESs) is often attributed to a preferentialexclusion/preferential-hydration mechanism, wherein osmolytes are excluded from the protein's surface. However, our understanding of the intricate relationship between biomolecular protection mechanisms in NADES and the role of water remains incomplete. We enhanced the accuracy of the General Amber Force Field (GAFF) for predicting properties such as density and viscosity for a betaine-glycerol DES, resulting in significant improvements with a minor 0.58% overestimation of density and a 7% underestimation of viscosity compared to experimental data. We delved into the structural behavior of ubiquitin, a small protein, within aqueous and Betaine-Glycerol-Water (Bet:Gly:Wat) (1:2:ζ; ζ = 0, 1, 2, 5, 10) DESs, exploring a temperature range from 298 K to 450 K. Our results reveal that (Bet:Gly) traps water molecules stopping them from forming a hydration layer around ubiquitin. This unique mechanism enhances protein stability in the DES compared to conventional theories used to explain osmolyte-mediated stabilization. Protein dynamics are closely tied (slaved) to solvent fluctuations. (Bet:Gly) slows down protein dynamics compared to water, preserving protein structures. These findings highlight the importance of proteinsolvent interactions and offer insights into non-monotonic protein folding in DESs with varying water levels, showing that protein unfolding in DESs only occurs at high temperatures. Finally, we explored how different DESs affect mammalian cells. Betaine-Glycerol-SucroseWater (2:3:1:5) shows unique cell volume behavior similar to glycerol and sucrose, hinting at a hybrid cryoprotective mechanism. Another DES, Trehalose-Glycerol (1:30) behaves like DMSO in terms of cell volume recovery but has a late-stage decrease. |
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| Autores principais: | Gomes, Inês Filipe |
| Assunto: | Solventes Eutéticos Profundos Dinâmica Molecular Solvatação Ubiquitina Criopreservação Teses de mestrado - 2024 |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Deep eutectic solvents (DESs) have gained prominence as promising alternative solvent media in biocatalysis and biomolecular (cryo)preservation for fragile biomolecules, such as DNA, RNA, cells (including stem cells), and even organs intended for transplantation. The stabilization of proteins by osmolytes, common components of natural DESs (NADESs) is often attributed to a preferentialexclusion/preferential-hydration mechanism, wherein osmolytes are excluded from the protein's surface. However, our understanding of the intricate relationship between biomolecular protection mechanisms in NADES and the role of water remains incomplete. We enhanced the accuracy of the General Amber Force Field (GAFF) for predicting properties such as density and viscosity for a betaine-glycerol DES, resulting in significant improvements with a minor 0.58% overestimation of density and a 7% underestimation of viscosity compared to experimental data. We delved into the structural behavior of ubiquitin, a small protein, within aqueous and Betaine-Glycerol-Water (Bet:Gly:Wat) (1:2:ζ; ζ = 0, 1, 2, 5, 10) DESs, exploring a temperature range from 298 K to 450 K. Our results reveal that (Bet:Gly) traps water molecules stopping them from forming a hydration layer around ubiquitin. This unique mechanism enhances protein stability in the DES compared to conventional theories used to explain osmolyte-mediated stabilization. Protein dynamics are closely tied (slaved) to solvent fluctuations. (Bet:Gly) slows down protein dynamics compared to water, preserving protein structures. These findings highlight the importance of proteinsolvent interactions and offer insights into non-monotonic protein folding in DESs with varying water levels, showing that protein unfolding in DESs only occurs at high temperatures. Finally, we explored how different DESs affect mammalian cells. Betaine-Glycerol-SucroseWater (2:3:1:5) shows unique cell volume behavior similar to glycerol and sucrose, hinting at a hybrid cryoprotective mechanism. Another DES, Trehalose-Glycerol (1:30) behaves like DMSO in terms of cell volume recovery but has a late-stage decrease. |
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