Micellisation and immunoreactivities of dimeric β-caseins

Bovine β-casein (β-CN) is a highly amphiphilic micellising phospho-protein showing chaperone-like activity in vitro. Recently, existence of multiple sequential epitopes on β-CN polypeptide chain in both hydrophilic–polar (ψ) and hydrophobic–apolar domains (φ) has been evidenced. In order to clarify specific contribution of polar and apolar domains in micellisation process and in shaping immunoreactivity of β-CN, its dimeric/bi-amphiphilic “quasi palindromic” forms covalently connected by a disulfide bond linking either N-terminal (C4 β-CND) or C-terminal domain (C208 β-CND) were produced and studied. Depending on the C- or N-terminal position of inserted cysteine, each dimeric β-CN contains one polar/apolar region at the centre and two external hydrophobic/hydrophilic ends. Consequently, such casein dimers have radically different polarities/hydrophobicities on their outside surfaces. Dynamic light scattering (DLS) measurements indicate that these dimeric casein molecules form micelles of different sizes depending on arrangement of polar fragments of the β-CN mutants in their constrained dimers. Non-aggregated dimers have different hydrodynamic diameters that could be explained by their different geometries. Measurements of fluorescence showed more hydrophobic environment of Trp residues of C208 β-CND, while in similar experimental conditions Trp residues of C4 β-CND and native β-CN were more exposed to the polar medium. Both fluorescence and DLS studies showed greater propensity for micellisation of the dimeric β-CNs, suggesting that the factors inducing the formation of micelles are stronger in the bi-amphiphilic dimers. 1-Anilino-naphthalene-8-sulfonate (ANS) binding studies showed different binding of ANS by these dimers as well as different exposition of ANS binding (hydrophobic) regions in the micellar states. The differences in fluorescence resonance energy transfer (FRET) profiles of C4 β-CND and C208 β-CND can be explained by differences of distances and/or by differences of relative orientations of the donor (Trp) and acceptor (ANS), as well as by differences in quenching properties of the disulfide bridges and intra-molecular hydrophobic interactions. The immunoreactivity assays showed somewhat lower IgE response to C208 β-CND than to C4 β-CND. Thus, dimerization of C208 β-CN, connecting two C-terminal hydrophobic domains of two monomers doubling long-range hydrophobic interactions, possibly may hide a part of epitopes in the hydrophobic interface/core of C208 β-CND that is consistent with the results of DLS and fluorescence studies. The obtained results indicate structural differences of dimers – possibly the formation of Y- and U-shaped structures for C208 β-CND and C4 β-CND, respectively. This study not only demonstrated the importance of the organization of polar and hydrophobic regions during micellisation of the constrained and oriented β-CN dimers but also confirmed a possible role of C-terminal hydrophobic domain in the immunoreactivity profile of native β-CN.