| Carbohydrates have been shown to increase the thermal stability of proteins. The mechanisms by which sugars increases stability, however, are not completely understood. I've used the combination of capillary electrophoresis (CE) and protein charge ladders, a collection of protein derivatives that differ incrementally in charge, to study the effects of fructose, and sucrose on the stability of RNase A and alpha-lactalbumin. This technique allows one to measure both the change in free energy of unfolding, Delta GN→D, and the change in hydrodynamic radius associated with unfolding in a single experiment. Knowing the difference between the hydrodynamic radius of the native and denatured states of the protein, one can calculate the contribution to the change in DeltaGN→D upon the addition of sugar due solely to excluded volume. I have shown that this effect, as calculated by the explicit water formulation of scaled particle theory (SPT), could entirely account for the change in Delta GN→D at 25°C caused by the introduction of carbohydrates in the cases studied. In addition I used SPT, and experimentally determined values of unfolded RNase A's RH, to calculate preferential binding parameters of sucrose to the denatured state of RNase A. This allows one to estimate the difference in number of sucrose molecules excluded from the denatured state compared to the native state. From this analysis we calculate that in the presence of 1M sucrose approximately 6-7 more sucrose molecules are excluded from the denatured state of RNase A compared to the native.; The effects of cosolutes on protein stability were further studied using simulations of a simple "HP" protein in the presence of solute molecules that are devoid of enthalpic interactions. It was found that entropic interactions were able to account for the increase in stability of our model protein as well as the exclusion of solute molecules from the protein surface. This simulation study also examined the impact of cosolute size and shape on its ability to stabilize the native state of the model protein.; At higher carbohydrate concentrations and elevated temperatures we experimentally observed the onset of aggregation. Thus while the sugar molecules stabilize alpha-lactalbumin against thermal denaturation, at sufficient concentrations they promote the aggregation of the denatured protein. In order to understand this phenomenon protein-protein interactions in this state have been modeled using the expressions for the potential of mean force derived by Prausnitz and Blanch. This model is able to capture the fact that protein interactions change from repulsive to attractive after the addition of sugar. Also despite its idealizations the model has been able to correctly predict the effects of changing the ionic strength of the solution and the effects of different sugars. |
