Modifications of the charges at the N-terminus of bovine β-casein: Consequences on its structure and its micellisation

Due to the combination of flexibility and amphiphilicity, in solution β-casein (β-CN) acts as a surfactant molecule. It undergoes self-association and forms “micellar-like” aggregates in aqueous media. The aim of our study was to understand the role played by the charges borne by the N-terminal part of β-CN in its association behavior. β-CN polarity was modified in order to induce new aggregation properties and to understand better the main factors defining them. An unphosphorylated wild type recombinant form (WT A2) of β-CN has been obtained by heterologous expression in the prokaryotic host Escherichia coli. A mutant (MU A2) form of β-CN was engineered by duplicating the first 6 N-terminal amino acids residues (RELEEL) of WT A2, three additional negatively charged glutamic acids replacing some of the negative charges lost by the absence of phosphorylation in prokaryotic expression system. Dynamic light scattering and fluorimetry experiments showed that recombinant and native β-CN have different behaviors in temperature-dependent association. Micellisation was impaired by the absence of phosphate groups. Mutation conferred to the protein the ability to form aggregates whose size is inversely proportional to the concentration. These different behaviors during the association processes displayed by recombinant β-CN are correlated with modifications of the temperature-induced compaction of the proteins, what was revealed by circular dichroism measurements. WT A2 was less condensed at the air-water interface than native β-CN; this was in part reversed by the additive mutation. However, MU A2 compensating the lack of phosphate charges in recombinant caseins forms a more cohesive monolayer at the air–water interface than do the other β-CN, which means that the mutation reinforces monomers interaction at the surface.