Unfolding of beta‐lactoglobulin on the surface of polystyrene nanoparticles: Experimental and computational approaches

Abstract

Structural changes ensuing from the non-covalent absorption of bovine beta-lactoglobulin (BLG) on the surface of polystyrene nanoparticles were investigated by using spectroscopic approaches, by assessing the reactivity of specific residues, and by limited proteolysis/mass spectrometry. Also, the immunoreactivity of absorbed and free BLG was compared. All these approaches indicated substantial rearrangements of the protein structure in the absorbed state, in spite of the reported structural rigidity of BLG. Changes made evident by experimental measurements were confirmed by computational approaches. These indicate that adsorption-related changes are most marked in the area between the main C-terminal alpha helix and the beta-barrel, and lead to full exposure of the thiol on Cys121 , consistent with experimental measurements. In the computational model of bound BLG, both Trp61 and Trp19 also move away from their neighboring quenchers and become solvent-exposed, as indicated by fluorescence measurement. Upon binding, the beta-barrel also loosens, with a substantial increase in immunoreactivity and with noticeable changes in the trypsinolytic pattern. The possible general significance of the structural changes reported here for non-covalently adsorbed BLG is discussed with respect to recognition events involving surface-bound proteins, as are aspects related to the carrier function(s) of BLG, and to its use as a common ingredient in many food systems.

Rubredoxin refolding on nanostructured hydrophobic surfaces: Evidence for a new type of biomimetic chaperones

“…Titration experiments with apoRd and subsequent separation of the unbound protein through Centricon devices were carried out to assess the amount of protein bound to 46‐nm NP. Under the conditions used in this part of the study (i.e., ≈ 50 micromolar apoRd and NP concentrations in the 2–5 g/L range), about 200 apoRd molecules were found to be adsorbed on the surface of each NP (not shown), consistent with that observed with other negatively charged proteins that interact with the NP surface through hydrophobic interactions. The number of bound molecules may appear low when considering an estimated “footprint” of ≈7 nm for the native protein, but it seems reasonable to expect this “footprint” to be sensibly larger in the case the protein unfolds on the NP surface, even without considering structural fluctuation and mutual repulsion among the bound proteins.…”

“…The requirement for apoRd contact with the NP surface in the “catalysis” of holoRd formation is underscored by the evidence presented in Figure , which compares the time courses of holoRd formation when 46 nm NP were used before and after pre‐coating with bovine beta‐lactoglobulin, a protein that tightly–but not covalently–adheres to NP through hydrophobic interactions . The precoating clearly impairs the NP's ability to function as a catalyst for holoRd formation.…”

“…Some physical properties of the NP are summarized in the Supporting Information Table S1. In some experiments, 46 nm NP were pre‐coated with a sub‐saturating amount of bovine beta‐lactoglobulin (0.05 g/L protein–equivalent to ≈0.003 m M –and 0.625 g/L NP) following procedures reported elsewhere …”

“…The requirement for apoRd contact with the NP surface in the "catalysis" of holoRd formation is underscored by the evidence presented in Figure 3, which compares the time courses of holoRd formation when 46 nm NP were used before and after pre-coating with bovine beta-lactoglobulin, a protein that tightly-but not covalently-adheres to NP through hydrophobic interactions. 30 The precoating clearly impairs the NP's ability to function as a catalyst for holoRd formation. This observation also provides circumstantial evidence of the stability of the interaction between NP and a relatively large (Mr 16000 Da) and hydrophobic protein, such as beta-lactoglobulin, that cannot be displaced from the NP surface by the much smaller and much less hydrophobic apoRd even when this latter is present in relatively large excess.…”

“…Other studies have demonstrated that several proteinsincluding proteins with remarkable structural stiffness, such as bovine beta-lactoglobulin-undergo large structural changes when allowed to interact with the hydrophobic surface of polystyrene nanoparticles (NPs) in the absence of chemical or physical denaturation. 30 The structural changes ensuing from adsorption resulted in the exposure of buried amino acid side chains. 31 Similar unfolding events have been shown to occur with proteins exposed to other types of hydrophobic nanoparticles, 32-35 and to be dependent on the geometrical features of the nanosystem.…”