Protein Hydration in a Bioprotecting Mixture

Abstract

We combined broad-band depolarized light scattering and infrared spectroscopies to study the properties of hydration water in a lysozyme-trehalose aqueous solution, where trehalose is present above the concentration threshold (30% in weight) relevant for biopreservation. The joint use of the two different techniques, which were sensitive to inter-and intra-molecular degrees of freedom, shed new light on the molecular mechanism underlying the interaction between the three species in the mixture. Thanks to the comparison with the binary solution cases, we were able to show that, under the investigated conditions, the protein, through preferential hydration, remains strongly hydrated even in the ternary mixture. This supported the water entrapment scenario, for which a certain amount of water between protein and sugar protects the biomolecule from damage caused by external agents.

Impact of the Environment on the PNIPAM Dynamical Transition Probed by Elastic Neutron Scattering

“…Preferential exclusion of stabilizing compounds is observed also in aqueous solutions of proteins. − In particular, an h dependence of T d similar to that shown in Figure has been observed for lysozyme embedded in water/glycerol and water/glucose matrices, where T d reaches its limiting value for h ≳ 0.4. − On comparing the two systems, it seems that preferential exclusion starts to take place at similar values of h in both PNIPAM and lysozyme, which suggests that PNIPAM linear chains have a capability to coordinate and confine water molecules comparable to proteins. This is likely an essential requisite for the “biomimic” character of PNIPAM, especially considering that it is precisely this kind of confined, proximal water that plays a major role in determining protein functional dynamics …”

Hydration Dynamics of Model Peptides with Different Hydrophobic Character

“…Structural information on hydration water can be obtained by analyzing the OHstretching band of the infrared spectrum, which is sensitive to modulations of hydrogen bonding interactions [53][54][55][56][57]. Figure 6 shows a comparison between the ATR-FTIR OHstretching bands obtained for NALMA and NAGMA solutions (75 mg/mL), and for the bulk solvent, composed by a mixture of H 2 O/D 2 O (10% w/w).…”

“…The band of the solvent is mostly due to the OH stretching modes of HOD species, and is not influenced by intraand inter-molecular vibrational coupling, thus facilitating the spectral interpretation [55]. The OH signal is sensitive to the strength of hydrogen bonds formed by the OH group (as proton donors): the formation of stronger H-bonds shifts the OH stretching distribution to lower frequencies (red-shift) [55,57,58].…”

The Role of Glycerol in Preserving Proteins Needs to Be Reconsidered

“…As an indispensable component of living organisms, proteins are widely used in biological reactions, enzyme engineering, molecular biology, and clinical treatment. , Due to the complexity and fragility of a protein’s structure, an enzyme often exhibits poor stability and biological activity in complex application environments, which limits its application in scientific research and biotechnology. Additives as protectants, such as salts, sugars, amino acids, and polyols, are usually added to increase protein stability. , In addition to the common mechanisms of action such as inducing correct protein folding and improving denaturation stability, − amino acid and polyol additives often have no effect on the functional activity of natural proteins. − Also, low concentrations of salts are able to prevent protein aggregation by increasing the solubility, and sugar protectors maintain the hydration shell of the protein. − However, the specific molecular mechanisms of protectants for inducing protein stability remain to be studied …”