Sucrose is preferentially excluded from surface of proteins (Timasheff Mechanism), increases protein chemical potential and stabilizes native state

To answer the quiz, you must understand the manner in which sucrose interacts with protein molecules in solution and the mechanistic consequences. Below, the quiz cases and the question are restated. In the posted slides, the preferential exclusion of sucrose (Timasheff Mechanism) and its mechanistic consequences are explained in detail. Here, I provide a very brief summary.

First, we need to consider the Wyman Linkage Function and the effects of strongly binding stabilizing ligands on protein stability. Ligand binding to the native protein state reduces the chemical potential of the protein. Because there is much more binding to native state than to the denatured state, there is much more reduction in the native state's chemical potential, and, thus, the free barrier between the states is increased; the native state is stabilized.

The illustration above shows a cartoon of the sucrose-protein interaction. Sucrose is preferentially excluded from the surface of proteins. That is, there is a lower sucrose concentration around the surface of the protein than in the bulk solution. This preferential exclusion mechanism was shown by our friend and mentor, Prof. Serge Timasheff. Preferential exclusion of a solute from the surface of the protein can be thought as "negative ligand binding", which increases chemical potential of a protein. This increase in chemical potential is the key aspect of the mechanism that helps explain the impact of sucrose in the two cases.

In case 1, the increase in protein chemical potential in the presence of sucrose would increase freeze-thawing induced reversible precipitation. The protein precipitates in this case because during freeze concentration the solubility limit of the protein is exceeded. Ice is formed from pure water, and the protein and other solutes are concentrated in the non-ice phase.

Upon thawing the precipitated protein native protein molecules are "diluted" and the protein molecule go back into solution. If sucrose were present in the solution, the increase in protein chemical potential would cause a decrease in protein solubility. As a result during freezing more protein would precipitate.

In case 2, preferential exclusion of the sucrose increases chemical potential of protein, but in this case this interaction inhibits freezing-induced irreversible aggregation. The magnitude of the preferential exclusion and the increase in chemical potential depends directly of the surface area of the protein state. The chemical potential increase is greater for the denatured than for the native state and, thus, the free energy barrier between the two states is increase (see cartoon above). Similarly, the free energy barrier between the most compact native conformations and partially unfolded, aggregation-competent species is increase in the presence of sucrose. Thus, sucrose inhibits stress-induced aggregation.

Two cases with monoclonal antibodies. For both, the bulk drug substances appeared cloudy after freeze-thawing. 

 Case 1. Upon thawing the bulk drug substance, the solution was very cloudy, due to protein precipitation. The precipitate could be collected by centrifugation, immediately after thawing. Infrared spectra of the centrifuged precipitate indicated that there was only native secondary structure in the protein molecules in the precipitate. After an uncentrifuged sample was held at room temperature for 2-8 hours, the precipitates dissolved and the protein molecules went back into solution. 

 Case 2. Upon thawing the bulk drug substance, the solution was very cloudy, due to protein precipitation. After the sample was held at room temperature for 2-8 hours, the precipitates settled, but did not go back into solution. Infrared spectra of the centrifuged precipitate indicated that there was extensive intermolecular beta sheet structure between the protein molecules in the aggregates.  I

In which case(s) would the inclusion of 0.3 M sucrose reduce the amount of precipitation; that is,  inclusion of the sugar in the bulk drug substance solution would be an effective formulation strategy?