Nanoparticle surface curvature influences protein stability and binding energetics

Nanotechnology has versatile applications in medicine, industry, and biotechnology. Tailoring nanoparticle interactions with physiologically relevant environments is a critical consideration in these fields. In particular, the protein corona that forms around nanoparticles is of high biomedical relevance because of its ability to alter nanoparticle behavior in vivo. Here, we report the effect of nanoparticle size on protein adsorption and unfolding stabilities. We examined two different proteins, R2ab and GB3, both bacterial surface proteins, and we analyzed their binding to polystyrene nanoparticles. For comparison, we also examined how these proteins bind to flat polystyrene plates. R2ab is known to bind strongly to polystyrene, leading to biofilm formation in S. epidermidis, while GB3 is an IgG binding protein used as a comparative control. Isothermal titration calorimetry allowed us to analyze binding parameters of both R2ab and GB3 binding to PSNPs, and association constants (K_a) values indicate tighter binding of R2ab and GB3 to smaller PSNPs. All the observed protein – nanoparticle interactions were enthalpically driven. Structural changes of the adsorbed proteins were assessed by both intrinsic tryptophan fluorescence and ANS fluorescence spectroscopy and cross-validated by far-UV circular dichroism spectroscopy. Both proteins appear to unfold when adsorbed, and the effect becomes weaker with increasing PSNP size. A comparison of the experimentally obtained folding ΔG° values for native proteins and nanoparticle-bound proteins indicates a destabilization of proteins on a surface. Consistent with structural studies, this destabilization becomes less pronounced for larger PSNPs. This work demonstrates the importance of side-on interactions between neighboring proteins, which can stabilize proteins bound to surfaces with low curvature. These interactions are likely important in the protein corona and could preserve the structure of surface-adsorbed proteins on nanoparticles in vivo.