Structural differences between bovine A¹ and A² β-casein alter micelle self-assembly and influence molecular chaperone activity

Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A¹ and A² β-casein (β-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A¹ and A² β-CN variants differ by a single AA, the substitution of histidine for proline at position 67. β-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A¹ and A² β-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A² β-CN variant formed smaller micelles than A¹ β-CN, with the monomer-micelle equilibrium of A² β-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 β-CN variants associated with the adoption of greater polyproline-II helix in A² β-CN and most likely led to enhanced chaperone activity of A² β-CN compared with A¹ β-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A¹ and A² milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as β-CN and, in this case, may have an effect on the functionality of milk.