The effect of C-terminal deletion on the folding and self-association of recombinant non-phosphorylated human β-casein

Recombinant human β-casein (CN) mutants were prepared having 11, 22 and 31 amino acids (aa) deleted from the C-terminus. The temperature-dependent self-association of these and the wild-type recombinant was studied by turbidity (OD_{400}) while possible folding differences were examined by intrinsic and extrinsic fluorescence intensity and fluorescence resonance energy transfer. There were major self-association and some conformational differences. Hydrophobicity profile and hydrophobic cluster analysis for bovine and human β-CN suggested that the ability of the 31 aa deletion mutant in human β-CN to self-associate when a comparable bovine deletion peptide would not may be due to the presence of additional hydrophobic regions in the middle, indicating that the human protein may contain more than a single hydrophobic binding locus and suggesting that the process for the formation and structure of the micelles of human milk may be quite different from that for bovine milk. A new model may be needed.