Preliminary Study on the Heterogeneous Nucleation Behavior and Interfacial Mechanism of Lysozyme Regulated by Silica Nanoparticles

Protein crystal nucleation remains difficult to predict and control and is still a critical issue in structural biology, protein crystal formulations, and crystal engineering. Heterogeneous nucleants can regulate protein crystallization by providing solid interfaces, enriching protein molecules, and stabilizing prenucleation aggregates; however, their dominant action stage, particle-size effect, and interfacial interaction mechanism remain unclear. In this study, hen egg white lysozyme (HEWL) was selected as a model protein, and silica nanoparticles (SNPs) with average diameters of 80, 120, and 200 nm were prepared using the modified Stöber method. Under a constant total particle surface area, the effects of SNPs on HEWL crystallization, prenucleation aggregation, interfacial adsorption, and subsequent crystal growth were systematically investigated. The results showed that, under 30 mg·mL−1 HEWL and 0.6 M NaCl conditions, all SNPs shortened the apparent induction time, with 200 nm SNPs showing the strongest effect. In contrast, 80 nm SNPs produced the largest crystal size at the fixed observation time of 72 h, suggesting that crystal size within a fixed incubation period is jointly affected by nucleation rate, nucleus number, local solute consumption, and subsequent crystal growth. Under low protein concentration and low NaCl concentration conditions, SNPs promoted crystal formation in systems with weak spontaneous nucleation. Zeta potential, UV-Vis, fluorescence, FT-IR, CD, and DLS results suggested that SNPs interacted with HEWL at the interface and promoted apparent aggregation behavior, whereas polarized optical microscopy indicated no detectable influence on the later linear growth of visible crystals. These results suggest that SNPs mainly affect processes before visible crystal formation and provide insight into their application as heterogeneous nucleants for protein crystallization.