Effect of self-association of αs1-casein and its cleavage fractions αs1-casein(136-196) and αs1-casein(1-197), on aromatic circular dichroic spectra:: comparison with predicted models

The self-association of native alpha(s1)-casein is driven by a sum of interactions which are both electrostatic and hydrophobic in nature. The dichroism of aromatic side chains was used to derive regio-specific evidence in relation to potential sites of alpha(s1)-casein polymerization. Near-ultraviolet circular dichroism (CD) revealed that both tyrosine and tryptophan side chains play a role in alpha(s1)-casein associations. Spectral evidence shows these side chains to be in an increasingly nonaqueous environment as both ionic strength and protein concentration lead to increases in the degree of self-association of the protein from dimer to higher oligomers. Near-UV CD investigation of the carboxypeptidase A treated peptide, alpha(s1)-casein(1-197), indicated that the C-terminal residue (Trp199) may be superficial to these interactions, and that the region surrounding Trp164 is more directly involved in an aggregation site. Similar results for the cyanogen bromide cleavage peptide alpha(s1)-casein(136-196) indicated the presence of strongly hydrophobic interactions. Association constants for the peptides of interest were determined by analytical ultracentrifugation, and also were approximated from changes in the near-UV CD curves with protein concentration. Sedimentation equilibrium experiments suggest the peptide to be dimeric at low ionic strength; like the parent protein, the peptide further polymerizes at elevated (0.224 M) ionic strength. The initial site of dimerization is suggested to be the tyrosine-rich area near Pro147, while the hydrophobic region around Pro168, containing Trp164, may be more significant in the formation of higher-order aggregates. (C) 1999 Elsevier Science B.V. All rights reserved.