Molecular Packing, Hydrogen Bonding, and Fast Dynamics in Lysozyme/Trehalose/Glycerol and Trehalose/Glycerol Glasses at Low Hydration

Abstract

Water and glycerol are well-known to facilitate the structural relaxation of amorphous protein matrices. However, several studies evidenced that they may also limit fast (∼picosecond-nanosecond, ps-ns) and small-amplitude (∼Å) motions of proteins, which govern their stability in freeze-dried sugar mixtures. To determine how they interact with proteins and sugars in glassy matrices and, thereby, modulate their fast dynamics, we performed molecular dynamics (MD) simulations of lysozyme/trehalose/glycerol (LTG) and trehalose/glycerol (TG) mixtures at low glycerol and water concentrations. Upon addition of glycerol and/or water, the glass transition temperature, T, of LTG and TG mixtures decreases, the molecular packing of glasses is improved, and the mean-square displacements (MSDs) of lysozyme and trehalose either decrease or increase, depending on the time scale and on the temperature considered. A detailed analysis of the hydrogen bonds (HBs) formed between species reveals that water and glycerol may antiplasticize the fast dynamics of lysozyme and trehalose by increasing the total number and/or the strength of the HBs they form in glassy matrices.

Trehalose or Sucrose: Which of the Two Should be Used for Stabilizing Proteins in the Solid State? A Dilemma Investigated by In Situ Micro-Raman and Dielectric Relaxation Spectroscopies During and After Freeze-Drying

“…Therefore, these original dielectric results in lysozyme þ disaccharide freeze-dried samples come in line with the scenario describing that some water molecules make bridges between the protein and the disaccharide. 9,62,63 A comparison between a HN parameter determined in Lys þ Treh and Lys þ Sucr, which characterizes the broadening of the relaxation peak, reveals that all values are lower in the former, indicating a broad distribution of the relaxation times (Fig. 5b).…”

“…It was shown by MD simulations that residual water has an antiplasticizing effect on the vitreous matrix by decreasing the size of free volume holes within the porous matrix and by strengthening H-bond interactions. 63 This effect is probably enhanced in Lys þ Treh sample because of the greater affinity of trehalose to water. This ability of trehalose to reduce this type of molecular mobility can be a specific property for preserving the protein conformation during long-term storage of the cake at room temperature, below T g .…”

Properties of Aqueous Trehalose Mixtures: Glass Transition and Hydrogen Bonding

“… 33 , 34 Water is thought to act as the anchor between the macromolecule and the glass matrix. 30 It was further suggested that higher viscosity of the trehalose solution results in hindered fluctuations of macromolecules. 35 , 36 Because of the significant difference in T g , the viscosity of trehalose mixtures remains higher than many other disaccharides and thus may enhance its protective effect.…”

“… 32 Yet, as was found for lysozyme in simulation, trehalose glass may be unable to fully replace all the hydrogen bonds that are available in diluted solution. 30 Beyond the direct macromolecule–sugar interactions, water–sugar hydrogen bonds may affect the glass matrix properties; for example, inelastic neutron scattering (INS) measurements show trehalose to be destructive to the water hydrogen bonding network. 37 In contrast, recent neutron diffraction experiments have shown little effect of trehalose on water structure.…”

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“…This antiplasticization role of small molecules has also been observed for glycerol (64,(128)(129)(130)(131)(132) and sorbitol (133) as well as in binary trehalose-water mixtures (26,40). Upon the addition of a small amount of glycerol, the T g of the lysozyme-trehalose(-water) system gradually decreases due to the plasticization effect of glycerol, but the molecular packing of the matrix embedding the protein improves in the presence of glycerol (132).…”