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A central paradigm that underpins our understanding of the interaction of proteins with solid surfaces is that protein adsorption leads to changes in secondary structure. The bound proteins tend to denature, and these non-native, adsorbed structures are likely stabilized through the loss of α-helices with the concomitant formation of intermolecular β-sheets. The goal of this work is to critically assess the impact this behavior has on protein desorption, where irreversible conformational changes might lead to protein aggregation or result in other forms of instability. The adsorption, desorption, and structural transitions of lysozyme are examined on fumed silica nanoparticles as a function of the amount of protein adsorbed. Surprisingly, the data indicate not only that adsorption is reversible but also that protein desorption is predictable in a coverage-dependent manner. Additionally, there is evidence of a two-state model which involves exchange between a native-like dissolved state and a highly perturbed adsorbed state. Since the in situ circular dichroism (CD) derived secondary structures of the adsorbed proteins are essentially unaffected by changes in surface coverage, these results are not consistent with previous claims that surface-induced denaturation is coverage dependent. Inspired by results from homopolymer adsorption experiments, we speculate that more local descriptors, such as the number of amino acids per chain that are physically adsorbed on the surface, likely control the desorption process.

Gaining more insight into the mechanisms governing the behavior of proteins at solid/ liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior.

Proteins usually fulfill their natural functions in the crowded cellular environment where they interact with a dense mixture of other biological macromolecules such as proteins. The concentration is very high, for example, up to several hundreds of mg/ml in cytosol of some E. coli, which is in contrast to the very dilute solutions used in most studies of function and structure. Here, we used Small-Angle Neutron Scattering (SANS) with the contrast matching technique to study the structure and oligomerization state of green fluorescent protein (GFP) in solutions containing various concentrations of the protein human serum albumin (HSA) as a crowding agent. By using HSA, a common protein in blood serum, we were creating a more biologically relevant crowding condition than other crowders usually used such as polyethylene glycol or Ficoll. GFP protein is a relatively stable protein with a beta-barrel structure, usually forming a dimer in dilute solution. By using perdeuterated GFP and hydrogenated HSA, we were able to probe only the GFP in the solutions by contrast matching HSA with an appropriate D2O/H2O buffer mixture. A series of HSA concentrations from 5mg/ml to 200 mg/ml were used. Analysis of the data indicates that GFP undergoes an HSA concentration-dependent transition that alters the way in which GFP oligomerizes in the solution. The dimer present in HSA-free solution remains unaltered by low concentrations of HSA. As the concentration of the crowder increases past 100 mg/mL, GFP adopts an ensemble of states with the transition to another dimeric configuration.

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