Calcium-Induced Changes to the Molecular Conformation and Aggregate Structure of β-Casein at the Air−Water Interface

Abstract

The influence of calcium on interactions of beta-casein at the air-water interface has been studied by several techniques, including interfacial rheology, atomic force microscopy (AFM), infrared reflectance-absorbance spectroscopy (IRRAS), and zeta potential measurements. In the absence of calcium, a weak interfacial gel forms after about 2.5 h. Also in the absence of calcium, the adsorbed beta-casein film exhibits some degree of both intra- and intermolecular structural organization. For example, IRRAS spectra show a measurable amount of alpha-helix content, and AFM images indicate the presence of interfacial aggregates with a characteristic lateral length scale of 20-30 nm, which we interpret as hemimicelles. Upon the addition of calcium, particularly at Ca:beta-casein molar ratios above approximately 5:1, a stronger interfacial gel forms more quickly; for example, the interfacial shear moduli increase twice as rapidly. Also under these conditions (5:1 Ca:beta-casein ratio) there is little evidence of structural organization; i.e., the alpha-helix peaks are very weak, and AFM images show a disordered, but continuous film, without distinct hemimicelles. On the basis of these findings, we hypothesize that calcium binding destabilizes the coupled intra- and intermolecular structural organization, and that the loss of organization permits more rapid interfacial gelation. These phenomena are characteristic of the air-water interface; they are not accompanied by analogous structural changes in bulk solution.

Rheological properties of protein films

“…However, the minimum protein concentration required for calcium ion induced aggregation can be considerably lower than for thermal aggregation of proteins such as the caseins. Vessely have studied Ca 2+ -induced aggregation of β-casein and monitored changes in the interfacial shear rheology [65] and the microscopic structure of the adsorbed β-casein films. The Ca 2+ ions induced a marked increase in η i that was associated with the formation of a network of more dense protein domains, describes as hemi-micelles.…”

External Factors Affecting Protein Aggregation

“…The proaggregation effects are generally mediated through binding to proteins. 256,257 Binding can result in small changes in tertiary structure in proteins, leading to formation of aggregation -competent conformers. For example, aluminum was found to bind to BSA, inducing protein aggregation through tertiary conformational changes, 167 and binding of aluminum to rhFVIII leads to both secondary and tertiary structural changes.…”

“…The hydrophobic property of air relative to water promotes protein adsorption at the interface, maximizing exposure of the hydrophobic residues to the air and potentially initiating aggregation. 257,347 Thus, agitiation may increase the amount of air -water interface that is available to facilitate surface -mediated unfolding and/or aggregation (see also Chapters 2 and 7 ). Stirring, without creation of new interfaces, can also increase the rate of mass transfer between bulk solution and existing interfaces, thereby increasing the turnover rate of protein adsorbing from bulk solution to interfaces between water and air or container -closure contacts.…”

Particle Formation and Aggregation of a Therapeutic Protein in Nanobubble Suspensions

“…22, 23 Conformational changes resulting from adsorption to the air-water interface have been associated with reduced efficacy in protein therapeutics. 24 Adsorption of protein particles to nanobubbles could result in protein particles and/or hybrid protein-air bubble nanoparticles, and potentially nucleate protein aggregation in the bulk solution.…”