Protein Crystallization from a Preordered Metastable Intermediate Phase Followed by Real-Time Small-Angle Neutron Scattering

We present a systematic study using real-time small-angle neutron scattering (SANS) and optical microscopy to follow the protein crystallization process in the presence of a metastable intermediate phase (MIP). Using bovine β-lactoglobulin (BLG) in the presence of the divalent salt CdCl₂ as a model system, we first determine the experimental phase behavior in D₂O. The protein solutions become turbid after crossing the first threshold salt concentration c*, and upon further increasing the salt concentration, the solutions become less turbid but not completely clear again. Thus, the second border is called pseudo-c**. Near pseudo-c**, crystallization follows a nonclassical process with a MIP, which is further explored with a focus on the structural evolution and the growth kinetics of the MIP prior to crystal nucleation. Real-time SANS measurements show that a correlation peak develops inside the MIP, and its peak position shifts to higher q-values with time, finally stabilizing at a characteristic length scale of d_MIP ≈ 84 Å. The area of this peak (proportional to the amount of MIP in the sample) increases with time first, reaches a maximum, and then decreases quickly upon crystallization due to consumption by crystal growth. The evolution of the correlation peak indicates a “preordering” nature of the MIP as precursors of crystal nucleation, which lowers the nucleation barrier for subsequent crystallization. These results of structural evolution and the role of MIPs during a nonclassical crystallization process may be relevant for other fields ranging from structural biology to pharmacy.