Effect of divalent cations and solubilizers in apoferritin and gamma d-crystallin solutions: Nucleation, crystallization and light scattering studies

Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding the processes involved in protein crystallization can help us gain a better understanding of such diseases. Furthermore, protein crystallization is necessary for protein structure resolution which is the first step towards determination of protein function. This is important since resolution of protein structure is the first step towards establishing structure/function relations, and possibly at performing specific structural modifications that may change the function in desirable directions. Another important application of protein crystallization is in downstream processing in the pharmaceutical industry where it is used for separation and as a final purification step. The present study increases knowledge of interactions between protein molecules during crystallization and hence the crystallization process.;Crystallization of a lens protein, human gamma D-crystallin (HGD), was studied in relation to cataract formation. The crystallization habits of this protein were determined in the presence of divalent cations which are found at elevated concentrations in cataractous lenses. Results indicate that the divalent cations studied enhance crystallization of HGD.;A thermodynamic property, the osmotic second virial coefficient, was measured in protein solutions and its value was correlated with the occurrence of crystallization. It was found that the second virial coefficient successfully predicted crystallization of HGD. A new method was developed for indirect measurement of the second virial coefficient using dynamic light scattering. This new method is more robust and efficient than the traditional static light scattering method.;Finally the ability of solubilizers to prevent crystallization of proteins was studied. A commercial solubilizer, NDSB-201, was found to increase the energy barrier to nucleation. Although this did not prevent crystallization, it resulted in fewer and smaller crystals being obtained. The naturally occurring alpha A-crystallin was a superior solubilizer to NDSB-201, as it suppressed aggregation and prevented crystallization of HGD under conditions for which NDSB-201 did not.;The findings in the present study provide insight into the processes by which protein crystallization occurs. Using the second virial coefficient to assess whether a protein will crystallize out of solution, approaches for retardation and prevention of protein crystallization, and implications for future research, are discussed.