Though proteins can be stored with cosolvents such as salts and polyols to enhance their stability and activity for biological and biotechnological applications, however, the mechanisms for protein stability remain elusive. Generally, these cosolvents promote the shifting of protein conformations towards a more compact form and prevent the association of the partially unfolded conformations that could lead to protein aggregation. Among those cosolvents, polyols are most widely used to stabilize proteins in aqueous solvents.
The protein stabilization and compaction of the tertiary structure of the protein promoted by polyols are mainly due to the excluded volume effect and solvophobic effect. These effects lead to the minimization of the solvent-accessible surface area of the protein conformation. However, by the same mechanism, polyols can also promote protein association reversibly. This is because the solvent-accessible surface area of the associated protein conformations is smaller than that of the unassociated proteins.
Glycerol is one of the most widely used polyols that is used to stabilize proteins. It is used in protein refolding, crystallization, and formulation of biopharmaceuticals. Glycerol when added to the protein, promotes protein compaction, and reduces protein flexibility. It also stabilizes the partially unfolded intermediates affecting both native and non-native protein aggregation.
For the study of the effect of glycerol in protein stabilization and prevention of protein aggregation, researchers used a hen egg-white lysozyme (HEL) dissolved in a glycerol/water mixture. During the study, it is found that the preferential interaction coefficient (ΓXP) value for a protein in a cosolvents/water mixture greatly varies among the distinct protein conformations.
Generally, the volume of the local domain of the protein increases with respect to the local domain of the protein in the crystal structure. Thus, an increment of the average volumes of solvent regions of the local domain of the protein after hydration is termed preferential hydration of volume increments (ΔVr). In this case, researchers found that the volume increment (ΔVr) is predominately occupied by solvent.
To further investigate the origins and consequences of significant change in the ΓXP, researchers characterized the effect of local protein conformational changes in protein stabilization. Using molecular dynamics simulation, researchers calculated ΓXP value and ΔVr of protein conformations.
Researchers also calculated the free energies of transfer (the transfer of free energy of the protein from pure water to an aqueous solution Δµp, tr) because the chemical potential (µp) of the protein changes when a cosolvent is added t an aqueous protein solution.
All these calculations, as mentioned before, indicate that the ΓXP value for simulation with fixed protein coordinates doesn’t significantly differ from that of the one in which protein backbone coordinates are constrained with respect to the reference structure. This indicates that the movement of the protein side chains doesn’t significantly affect the value of ΓXP. However, the conformational changes that can affect the ΓXP value of protein occur rapidly and are transient. This is in good agreement with the large transient local conformational changes as illustrated by hydrogen exchange experiments in previous research.
However, the structural heterogeneity between the protein conformations in 30 % v/v aqueous glycerol doesn’t clearly differ from the structural heterogeneity of protein conformations in pure water. This indicates that the effect of glycerol on the protein conformational sampling can’t be measured directly by the molecular dynamics simulations. Therefore, researchers used preferential interaction theory to understand the effect of glycerol on protein conformations after investigating the effect of conformational changes in the protein backbone that leads to the changes in the ΓXP.
In simulation one, conformational changes in the loop regions of the protein affect the ΓXP value. This is because initially, no glycerol molecule is accessible to N and O atoms for H-bonding. However, after 30 nanoseconds, loops of the protein open up leading to the formation of a cleft in between the loop and the rest of the protein. This leads to the glycerol molecules (up to 3 molecules) accessible to the N and O atoms of the protein side chains that are located inside the cavity.
Inside the cleft, one glycerol molecule resides for 8 nanoseconds and forms multiple H-bonding with N and O atoms of protein side chains. This is called glycerol-binding loci where solvent regions positively contribute to the increase in ΓXP. However, in simulation 4, there is no such specific interaction and also fewer protein N and O atoms are available for the multiple H-bonding with glycerol. This concludes that the conformational changes in the same loop region may or may not result in the exposure of the glycerol-binding loci at the protein surface
The preferential hydration of volume increments of the local domain reveals that the volume increments of the local domains are hydrated leading to the change in ΓXP value. Expanded protein conformations have lower ΓXP as compared to that compact conformations.
For one simulation with unconstrained coordinates, conformational changes with respect to the crystal structure are limited and the corresponding ΓXP value doesn’t change with respect to that of the crystal structure. However, for other unconstrained simulations, conformational changes in the loop regions are considerable and corresponding ΓXP values are also significantly lowered as compared to that of the crystal structure. This is because there is a significant increase in the number of water molecules in the local domains leading to an increase in the radial distances and a decrease in the degree of preferential hydration.
In conclusion, with the combination of the thermodynamic framework of preferential interactions with a molecular-level insight into the solvent-protein interaction, researchers have derived mechanisms of glycerol-induced protein stabilization. According to this mechanism, a series of 40 ns molecular dynamics simulations for the hen egg-white lysozyme in aqueous glycerol have shown conformational changes in the protein backbone that result in significant differences in the preferential interaction coefficient.
The two mechanisms underlying such differences are as follows;
1) The creation or deletion of the glycerol-binding loci at the protein surface that is contributed by a specific orientation of protein N and O atoms favoring the formation of multiple H-bonding with glycerol
2) The preferential hydration volume increments of the local domain near the protein surface.
The first mechanism is responsible for the specific changes in the protein energy surface while the second mechanism is responsible for the overall shift of the native protein promoting towards the more compact conformations. Combining these two mechanisms, protein compaction and stabilization by polyols are governed by the molecular size of the polyols and glycerol-induced protein stabilization/compaction mainly originates from the electrostatic interaction that leads to the orientation of glycerol molecules at the protein surface in such a way that glycerol is further excluded.
Furthermore, glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing the aggregation-prone partially unfolded conformations through the preferential interactions with hydrophobic surface regions that favor the amphiphilic interface orientation of glycerol. Preferential interactions with hydrophobic surface regions also occur for some other cosolvents such as urea and arginine, however, these cosolvents also preferentially interact with hydrophobic surface regions of the native protein because of that they don’t stabilize the native protein.
Reference article: Biochemistry (2009) doi: 10.1021/bi900649t