Effects of disulfide bridges and backbone connectivity on water sorption by protein matrices

Abstract

Understanding the water sorption behavior of protein powders is important in applications such as the preservation of protein-based pharmaceuticals. Most globular proteins exhibit a characteristic sigmoidal water adsorption isotherm at ambient conditions. However, it is not well understood how water sorption behavior is influenced by intrinsic factors that are related to structural properties of proteins. We investigate computationally how structural constraints on proteins influence the water sorption isotherms of amorphous protein powders. Specifically, we study the effects of non-local disulfide linkages and backbone connectivity using pheromone ER-23 and lysozyme as model proteins. We find that non-local disulfide linkages can significantly restrict structural changes during hydration and dehydration, and this in turn greatly reduces the extent of hysteresis between the adsorption and desorption branches. Upon removing the backbone connectivity by breaking all peptide bonds in lysozyme, we find that the hysteresis shifts towards the lower humidity regime, and the water uptake capacity is significantly enhanced. We attribute these changes to the higher aggregation propensity of the constraint-free amino acids in dehydrated condition, and the formation of a spanning water network at high hydration levels.

Protein Hydration Thermodynamics: The Influence of Flexibility and Salt on Hydrophobin II Hydration

“…As the protein becomes more flexible, its RMSD increases, and is accompanied by a concomitant increase in Δ G . Our finding that a rigid protein is hydrated more favorably is also consistent with the work of Debenedetti and co-workers, who demonstrated that the thermodynamic drive for water sorption by protein crystals increases when the rigidity of the proteins in the crystal is enhanced, either artificially or physically via disulfide bonds. , …”

“…We find that a protein can be hydrated more favorably by enhancing its rigidity; for example, by engineering it to have a higher content of rigidifying residues, like proline, − a high concentration of strong interactions, like salt bridges and disulfide bonds, ,,− or through chemical cross-linking. − Conversely, enhancing protein flexibility would serve to lower its propensity to remain hydrated, and in principle, it could not only lower protein solubility, but also be used to trigger protein assemblies. Intraprotein interactions, such as disulfide bonds or salt bridges, are also used to stabilize the folded state of a protein. ,, In addition to stabilizing the folded state directly, our findings suggest that such favorable interactions ought to also favor the folded state indirectly through their role in enhancing protein rigidity.…”

“…Our finding that a rigid protein is hydrated more favorably is also consistent with the work of Debenedetti and co-workers, who demonstrated that the thermodynamic drive for water sorption by protein crystals increases when the rigidity of the proteins in the crystal is enhanced, either artificially or physically via disulfide bonds. 10,11 ■ THE ROLE OF SALT CONTENT Most proteins can be rendered insoluble by the addition of certain salts; such a "salting out" of proteins is a commonly employed protein purification technique. 2,6 However, the phenomenon of salting-out depends on the nature of both the protein and the salt, and a molecular level understanding of specific ion effects in protein solubility, the so-called Hofmeister effects 7,75 remain elusive.…”

“…Thus, being able to efficiently estimate Δ G is an important first step toward better understanding protein solubility, and modulating Δ G offers a means to influence protein solubility. The hydration free energy of a protein also sheds light more directly on protein dehydration and rehydration processes, which are important for the storage of biologics as freeze-dried powders and for their subsequent reconstitution into therapeutics. − …”

Structure-transport relationships of poly(aryl piperidinium) anion-exchange membranes: Eeffect of anions and hydration

“…[60][61] In desorption mode, the bound water molecules keep the polymer chains apart, and the contractive forces are not sufficient to force the water molecules from the swollen structure as the water vapor pressure decreases, resulting in higher water content compared to that in sorption mode. The large sorption hysteresis was also found in weak polyelectrolytes, and other amorphous materials (e.g., protein powder), [62][63][64] in which case the sorption hysteresis was attributed to the polymer's resistance to plasticization by water (i.e., stiffer chains) due to its higher shear modulus and the larger stress relaxation time consequently hampers the response to RH changes. 65 While water content (λ) is used to describe the local hydration of ionomer moieties, water volume fraction,𝜙 𝑤 , is a better representation of the network-level properties.…”