Bridging the Gap in Cryopreservation Mechanism: Unraveling the Interplay between Structure, Dynamics, and Thermodynamics in Cryoprotectant Aqueous Solutions.

Cryoprotectants play a crucial role in preserving biological material, ensuring their viability during storage and facilitating crucial applications such as the conservation of medical compounds, tissues, and organs for transplantation. However, the precise mechanism by which cryoprotectants modulate the thermodynamic properties of water to impede the formation and growth of ice crystals, thus preventing long-term damage, remains elusive. This is evident in the use of empirically optimized recipes for mixtures that typically contain DMSO, glycerol, and various sugar constituents. Here, we use terahertz calorimetry, Overhauser nuclear polarization, and molecular dynamics simulations to show that DMSO exhibits a robust structuring effect on water around its methyl groups, reaching a maximum at a DMSO mole fraction of XDMSO = 0.33. In contrast, glycerol exerts a smaller water-structuring effect, even at higher concentrations (Scheme 1). These results potentially suggest that the wrapped water around DMSO's methyl group, which can be evicted upon ligand binding, may render DMSO a more surface-active cryoprotectant than glycerol, while glycerol may participate more as a viscogen that acts on the entire sample. These findings shed light on the molecular intricacies of cryoprotectant solvation behavior and have potentially significant implications for optimizing cryopreservation protocols.

Collective hydration dynamics of guanidinium chloride solutions and its possible role in protein denaturation: a terahertz spectroscopic study.

The remarkable ability of guanidinium chloride (GdmCl) to denature proteins is a well studied yet controversial phenomenon; the exact molecular mechanism is still debatable, especially the role of hydration dynamics, which has been paid less attention. In the present contribution, we have addressed the issue of whether the collective hydrogen bond dynamics of water gets perturbed in the presence of GdmCl and its possible impact on the denaturation of a globular protein human serum albumin (HSA), using terahertz (THz) time domain spectroscopy (TTDS) in the frequency range of 0.3-2.0 THz. The collective hydrogen bond dynamics is determined by fitting the obtained complex dielectric response in a multiple Debye relaxation model. To compare the results, the studies were extended to two more salts: tetramethylguanidinium chloride (TMGdmCl) and sodium chloride (NaCl). It was concluded that the change in hydration dynamics plays a definite role in the protein denaturation process.

Short chain polyethylene glycols unusually assist thermal unfolding of human serum albumin.

In the present study we have investigated the thermal stability of the globular transport protein human serum albumin (HSA), in the presence of two small chain polyethylene glycols (namely PEG 200 and PEG 400). Both near- and far-UV circular dichroism (CD) study reveal that addition of PEG moderately increases the α-helical content of the protein without abruptly changing its tertiary structure. The hydration structure at the protein surface experiences a notable change at 30% PEG (v/v) concentration as evidenced from compressibility and dynamic light scattering (DLS) measurements. Thermal denaturation of HSA in the presence of PEG has been studied by CD and fluorescence spectroscopy using the intrinsic fluorophore tryptophan and it has been found that addition of PEG makes the protein more prone towards unfolding, which is in contrary to what has been observed in case of larger molecular weight polymers. The energetics of the thermal unfolding process has been obtained using differential scanning calorimetry (DSC) measurements. Our study concludes that both the indirect excluded volume principle as well as interaction of the polymer at the protein surface is responsible for the observed change of the unfolding process.