Interaction of Beta-Casein with Kappa-Casein Fibrils

I. Portnaya, S. Avni, E. Kesselman, U. Cogan, D. Danino
Technion –Israel Institute of Technology,

Keywords: kappa-casein, fibrils, beta-casein, interaction


Amyloid protein fibrils are associated with numerous degenerative diseases. Kappa casein (kCN) at physiological conditions is known to form amyloid-like fibrils. Although these are not considered disease-related, understanding fibrillization and inhibition of this process may assist studying fibril formation phenomena in these diseases. Therefore, the associative behaviour of kCN, especially fibrillization, and ways to suppress it are currently of great interest. Recently, the possibility of inhibition of kCN fibrillization by another milk protein, beta-casein (βCN), was suggested. The mechanism of this impact is not yet clear. βCN is characterized by a highly polar, negatively charged N-terminal domain and a highly nonpolar C-terminal domain, and displays a pronounced self-association behavior. Micellization of βCN is a reversible process depending on temperature and pH. kCN is also amphiphilic protein, possessing a predominantly hydrophilic C-terminal block and a hydrophobic N-terminal end. However, kCN self-association has been found to be a more complex process. This protein contains two cysteine residues (Cys11 and Cys88). In the native form of kCN, intra- and inter-molecular disulfide bonds lead to the formation of multimeric species ranging from monomers to decamers, followed by further association of the subunits into micelles. In contrast, kCN, in which the disulfides were reduced (R-kCN), exhibits a monomer-micelle equilibrium. Both native kCN and R-kCN are known to also form fibrils. Their tendency to fibrillization increases with increasing of temperature and time of incubation, and R-kCN forms fibrils much more readily than native kCN. Unlike the micellar aggregates, fibril formation is irreversible. To elucidate the mechanism of inhibiting fibrils formation we studied the interaction between kCN (in its native and reduced forms) with βCN micellar solutions at different temperatures and incubation periods. There are two modes resulting in inhibition of kCN fibrillization. The first mode is mixed micellization, which is effective mainly at low temperatures and short incubation periods, notably in presence of sufficient kCN monomers. Our recent study is focused primarily on the second mode, which is more pronounced in presence of a considerable number of the fibrils. By combining time-resolution studies using Isothermal Titration Calorimetry and cryogenic-Transmission Electron Microscopy, with Small-Angle X-Ray Scattering and Fluorescence Microscopy we revealed the ability of βCN species (monomers and micelles) to be adsorbed on already existing fibrils. The adsorption of βCN on the fibrils inhibits their subsequent growth. The current finding may be important also for potential application of kCN fibrils for proteins adsorption in novel biomaterials.