The regulation of protein crystallization using choline chloride precipitant and its mechanism

Abstract

Inorganic salts are the most commonly used precipitants in protein crystallization. However, due to their strong ability to destroy the hydration shell and diffuse electric double layer of proteins, the use of inorganic salts can easily cause protein precipitation. This work studied protein crystallization using organic salt choline chloride (cc) as the precipitant. Compared with NaCl, crystallization, it was found that lysozyme could nucleate at a lower supersaturation and the single-crystal area was significantly enlarged with cc. Even at a high supersaturation of nearly 30, lysozyme crystals could still maintain a complete tetragonal morphology with no urchin-like crystals in cc solution. Furthermore, the average crystal size in cc solution was larger than that in NaCl solution even at a lower supersaturation. In addition, the quantitative efficiency of yield was about equal in cc and NaCl system. Combining Zeta potential, fluorescence spectrum and MD simulation, it was found that cc could interact with lysozyme through hydrogen bond, which weakened the damage of Cl− to diffuse electric double layer of proteins and increased the stability of lysozyme. DLS revealed that the presence of cc could promote the formation of large protein aggregates, which was favorable for crystal growth. In addition, CD spectra and lysozyme activity showed that the presence of cc did not affect the secondary structure and activity of lysozyme. This work proved that cc could be used as a substitute precipitant to grow protein crystals with large size and improve the stability of protein crystallization.