The distinct binding modes of pesticides affect the phase transitions of lysozyme

Abstract

Studying the aggregation and nucleation of proteins in the presence of organic molecules is helpful for disclosing the mechanisms of protein crystallization. In this work, taking into account the hydrophobicity of trifluoro-containing molecules, the effects of two pesticides, chlorfenapyr (CHL) and picoxystrobin (PIC), on the aggregation and crystallization of hen egg-white lysozyme (HEWL) were studied via the hanging-drop method. Upon characterization via dynamic light scattering (DLS), Raman studies, PXRD, etc., the results indicate that in the presence of low levels of CHL or PIC (30 μM), the nucleation of HEWL is enhanced; whereas at high additive levels (100 μM), both crystalline and amorphous phases are generated. The sizes of the protein aggregates increase almost linearly during the first 30 min of crystallization time. Without pesticide additives, the size growth rate is about 81.3 ± 11.1 nm min⁻¹, while in the presence of 30 μM CHL or PIC, the size growth rate rises to 94.2 ± 16.5 or 88.1 ± 18.8 nm min⁻¹, respectively. UV-vis, fluorescence, and circular dichroism spectroscopy studies were employed to disclose the binding mode and conformation changes of the HEWL–CHL and HEWL–PIC complexes, in combination with DFT calculations. Molecular simulations confirm that HEWL–CHL is dominated by non-polar interactions, resulting in protein molecules with larger hydrophobic surface areas, while HEWL–PIC forms more stable hydrogen bonds. These results suggest that the presence of small amounts of pesticides can lead to interactions strong enough to influence the aggregation and nucleation of the protein, which will affect the formation of protein crystals in competition with the amorphous fiber-like precipitates.