Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

Abstract

The equilibrium dissociation of recombinant human IFN-␥ was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was ؊209 ؎ 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 ؎ 1.6 nm 2 /molecule of dimer. The first-order aggregation rate of recombinant human IFN-␥ in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were ؊41 ؎ 9 ml/mol of dimer and 3.5 ؎ 0.2 nm 2 / molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values Ϸ4-fold larger for both transitions.A ggregation is of considerable concern to the medical, pharmaceutical, and biotechnology industries, as protein aggregation occurs in human diseases (1-4) and during the production, purification, and storage of protein products (5). Characterization of the conformational state(s) that lead(s) to aggregation is essential for a mechanistic understanding of the aggregation process.Protein aggregation was once viewed as a nonspecific hydrophobically driven process involving the fully unfolded random coil (6-8). However, recent research has shown protein aggregation to follow distinct pathways involving folding intermediates (6, 7, 9). Thus, the folding pathway and aggregation processes are critically linked (7,8). Characterization of the unfolding process, in conjunction with aggregation rate studies, should then provide insight into the mechanisms of protein aggregation and the role of folding intermediates therein. A major challenge in characterizing aggregation pathways is that under conditions that greatly favor the native state (e.g., physiological), the small populations of highly reactive aggregationcompetent species and the transition states leading to these species may be inaccessible to available spectroscopic techniques. To populate putative aggregation-competent species at levels sufficient for spectroscopic measurement, conditions that greatly perturb the native state are typically used (e.g., pH Ͻ4.0). Furthermore, even if the aggregation-competent species populated under nonn...

Estimating hydration changes upon biomolecular reactions from osmotic stress, high pressure, and preferential hydration experiments

“…This estimation was suggested to be consistent with the change in buried surface area (4,16). Since then, OSA has been applied to various biomolecular processes, including ion channels, DNA-protein, and carbohydrate-protein interactions (5)(6)(7)(8)(9)(10).…”

“…Osmotic stress analysis (OSA) aims to estimate the number of water molecules adsorbed (or released) as a result of biomolecular processes (4)(5)(6)(7)(8)(9)(10). To do so, osmolytes (such as glycerol and polyethylene glycol, known as protein stabilisers) are added to the system (4-6).…”

“…It was also suggested that OSA is based on a misinterpretation of the equation of preferential hydration (12,15). In addition, the number of water molecules enumerated by OSA often depends on the choice of osmolytes (9,10,12,13,17). This suggests that osmolytes do not always behave as assumed.…”

“…Partial molar volumes can be measured from high pressure experiments (19,20), as well as densimetry (18). The number of water molecules may be inferred by the use of a two-state model for water (21), or through the estimated water density increment in the vicinity of biomolecules (10,19).…”

“…The number of waters estimated from OSA and from volumetric analyses is often inconsistent (10,18,19,22). For example, a 4-fold difference was seen in the equilibrium dissociation of human IFN-␥ (10) and up to a 3-fold difference in the camphor binding of cytochrome P-450 (19).…”