Role of Specific and Nonspecific Interactions in the Crystallization Behavior of BSA and HSA Protein Solutions

The crystallization conditions of proteins are sensitive to the prevailing interactions. Even the two similar proteins, bovine and human serum albumin (BSA and HSA), exhibit different crystallization conditions despite their comparable function, biophysical properties, shape, and size (≈60 kDa and a 75.8% sequence identity). In this work, we provide a comparison of specific and nonspecific interactions regarding the crystallization behavior of BSA and HSA. The results of the analysis of crystal packing interfaces indicate that HSA uses a relatively larger part of its surface area to establish crystal contacts compared to its bovine counterpart. Likewise, HSA utilizes more of its residues for crystal contact formation, offering a broader range of options to establish attractive interactions. Phase diagrams of the BSA–PEG and HSA–PEG systems were established in order to gain more precise insights into the nonspecific depletion interactions. It turns out that BSA crystallizes predominantly via depletion interactions, whereas HSA does not. Subsequent systematic small-angle scattering (SAXS) measurements of the two systems in combination with quantitative modeling provide insights into the induced effective interactions, allowing for a better understanding of the two protein–PEG systems. The results obtained were compared to the previously established reentrant condensation (RC) phase behavior of BSA and HSA. The RC phase behavior is caused by the specific interaction of proteins with added multivalent cations. In this case, HSA crystallizes, but BSA does not. This comparison emphasizes the different roles of specific and nonspecific interactions for the crystallization behavior of BSA and HSA.

This study investigates the different crystallization behavior of the two similar proteins BSA and HSA. While trivalent salts induce specific interactions and promote HSA crystallization involving large areas of its crystal surface, BSA crystallization is promoted by nonspecific interactions via the addition of nonadsorption polymers. Phase diagrams, SAXS measurements, and crystal analyses provide a comprehensive description.