Investigating the Role of Surface Hydrophobicity in Protein Aggregation

Hydrophobic interactions between protein molecules are considered to be a significant contributor to attractive protein-protein interactions (PPIs) in solution. Attractive PPIs play critical roles in self-association and aggregation, thus affecting the overall protein stability. Surface hydrophobicity of three model proteins was characterized by hydrophobic interaction chromatography and fluorescence spectroscopy. To compensate for known limitations of these two widely used methods, a novel approach, based upon Nuclear Magnetic Resonance Spectroscopy (NMR), was investigated as a potential alternative. The degree of decrease in the transverse relaxation time (T2) of small molecule probe, such as phenol, due to its interaction with the protein of interest, was monitored to reflect the surface hydrophobicity. Utilization of this multi-method approach emphasized the differences in surface hydrophobicity of the three proteins and to distinguish the effects of two types of hydrophobic amino acids, aromatic and aliphatic, on surface hydrophobicity. Protein unfolding, interactions and aggregation mediated at the air/water interface were monitored. It was found that aggregation was not induced by mechanical stress for the studied proteins. Furthermore, the propensities to unfold or interact with the air/water interface were only influenced by the changes in pH and not by the degree of surface hydrophobicity. Building upon the knowledge gained from the three model proteins, the surface hydrophobicity of three unknown monoclonal antibodies (MAbs) was characterized and aggregation was monitored under mechanical stress at different ionic strength conditions. Our findings suggest that even when attractive interactions are significant, as in the case for MAb Y, the surface hydrophobicity alone is not the major factor affecting protein aggregation. Further, antibody aggregation was studied under thermal stress. Upon heating the MAbs, unfolding and the increase in their aggregation was observed. Additionally, the aggregation propensity of MAb Y was subjected to a combination of mechanical and thermal stresses, and it was found that the aggregation increased when more energy was applied to stress the protein. These results demonstrate that the hydrophobicity of a protein molecule is highly dependent on solution conditions and conformational changes of the protein. Therefore, protein surface hydrophobicity alone cannot be directly related to the protein propensity to aggregate and a combination of both the average and surface hydrophobicity should be taken into account