Hydrophobic Surface-Induced Protein Nucleation on Silicone Oil-Grafted Coverslips

Efficient protein crystallization remains a major requirement for structural studies based on X-ray diffraction. Surface-mediated nucleation has emerged as an effective strategy for promoting crystal formation, particularly under conditions where spontaneous nucleation is limited. In this work, silicone oil-grafted coverslips (SOC) were evaluated as hydrophobic substrates for protein crystallization using the vapor-diffusion method. The crystallization behavior of lysozyme, ferritin, and proteinase K on SOC was compared with that on nonsiliconized and commercially siliconized coverslips. Contact-angle measurements confirmed the hydrophobic nature of SOC, with a water contact angle of approximately 105°. The influence of SOC on crystallization was found to be protein dependent. Lysozyme exhibited the strongest response, particularly at low protein concentrations, where extensive crystal formation occurred on SOC while crystallization was absent or limited on the reference surfaces. Ferritin showed a marked increase in nucleation density, resulting in extensive microcrystallization, whereas proteinase K displayed a more moderate response. Raman spectroscopy revealed no significant changes in the characteristic secondary-structure bands of the resulting protein crystals, indicating preservation of the overall protein structure during crystallization on SOC. The results suggest that silicone oil-grafted surfaces primarily influence the nucleation stage of protein crystallization while maintaining the structural integrity of the resulting crystals. The observed effect was protein dependent and most pronounced under conditions where nucleation was otherwise limited. These findings highlight the potential of silicone oil-grafted interfaces as simple nucleation-promoting substrates for protein crystallization and structural biology applications.