HETEROGENEOUS NUCLEATION OF PROTEIN CRYSTALS MEDIATED BY NANOSTRUCTURED MATERIALS

X-ray diffraction crystallography is recognized as the predominant technique for elucidating the three-dimensional structure of macromolecules, especially proteins. The intrinsic correlation between the three-dimensional structure of proteins and their biological functions is well established, with the atomic arrangement being crucial for the conformation and, consequently, the activity of the protein. However, obtaining high-quality protein crystals remains a significant challenge due to the empirical process of crystallization, which is dependent on trial and error and involves high costs. Recently, heterogeneous nucleation using minerals and nanostructured materials has emerged as a promising approach to improve this process. These materials, with high surface areas and ability to form porous networks, can optimize crystallization conditions. The heterogeneous topography can provide a suitable environment for the crystallization of different proteins, possibly due to the formation of pockets between the particles, which provide nucleation sites for the initiation of crystal formation. Among the nanoparticles, metal-organic frameworks (MOFs) offer various surface topographies, pore sizes, and length scales. In this study, the crystallization of proteins in the presence of zeolitic imidazolate frameworks (ZIFs), a subclass of these materials, was investigated. ZIF-8 was chemically synthesized with zinc nitrate hexahydrate and 2-methylimidazole in the presence of methanol, and part of it was treated with ultrasonic bath to create defects in its structure. The morphology and chemical composition of both samples were characterized. Afterward, the nanoparticles were applied to protein solutions to study crystallization via sitting drop vapor diffusion. The presence or absence of crystals was observed using an automatic imager. The study also evaluated the interference of graphene oxide (GO) derivatives in the protein crystalline network through synchrotron X-ray diffraction. The results demonstrated that the introduction of treated nanomaterials significantly promoted the crystallization process compared to the trials with untreated nanoparticles and the control (without nanoparticle addition). Additionally, analyses shown that there was no significant interference in the diffraction of the protein crystals, revealing promising potential for future applications, although each material needs to be individually assessed. This work was supported by the São Paulo Research Foundation (FAPESP) under grant number 2023/17742-1.