Few experimental techniques allow the analysis of the protein corona in situ. As a result, little is known on the effects of nanoparticles on weakly bound proteins that form the soft corona. Despite its biological importance, our understanding of the molecular bases driving its formation is limited. Here we show that hemoglobin can form either a hard or a soft corona on silica nanoparticles depending on the pH conditions. Using cryoTEM and synchrotron-radiation circular dichroism, we show that nanoparticles alter the structure and the stability of weakly bound proteins in situ. Molecular dynamics simulation identified the structural elements driving protein-nanoparticle interaction. Based on thermodynamic analysis, we show that nanoparticles stabilize partially unfolded protein conformations by enthalpy-driven molecular interactions. We suggest that nanoparticles alter weakly bound proteins by shifting the equilibrium towards the unfolded states at physiological temperature. We show that the classical approach based on nanoparticle separation from the biological medium fails to detect destabilization of weakly bound proteins, and therefore cannot be used to fully predict the biological effects of nanomaterials in situ.

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