Protein Denaturation at the Air-Water Interface in the Context of Nanoparticle Soft Corona Studies

The interaction of proteins with surfaces can involve both protein binding and unfolding and has been investigated since the 1960s. Proteins in solution can adsorb onto and denature at both solid-liquid and gas-liquid interfaces, and the spontaneous adsorption of proteins on a nanoparticle (NP) surface to form a corona can be described as a specific case of adsorption at the solid-liquid interface, where the surface curvature also becomes important. While nanoparticles offer a large surface area, they are not the only surface in the sample with which the proteins interact due to the creation of other interfaces in an experimental setup. In this work, we examine the role of the air-water interface in protein denaturation via soft corona studies with citrate-capped gold nanoparticles. Chymotrypsin (ChT) protein in the soft corona was observed to exhibit an inverse relationship between concentration and denaturation, an air-water interface-mediated trend that, without the right control experiments, can be misinterpreted to be caused by protein-NP interactions. We show that any protein denaturation observed comes from the synergistic effect of the air-water interface and agitation and that NPs did not denature ChT in the "soft corona" in the absence of these two factors. While the propensity to adsorb and denature at hydrophobic interfaces varies among proteins with different structural properties, it is important to use simplified procedures that minimize interfacial areas other than the nanoparticle surface in corona studies to prevent misattribution of the observed effects.