Interactions of triply phosphorylated human β-casein: Fluorescence spectroscopy and light-scattering studies of conformation and self-association

Structural changes of triply phosphorylated human β-casein, caused by shifts in temperature between 5 and 40 °C, were studied using intrinsic and extrinsic fluorescence, fluorescence polarization, turbidity, and light scattering measurements. Intrinsic fluorescence declined between 5 and 20 °C then rose between 25 and 40 °C, indicative of a shift of the tryptophan fluor toward a more nonpolar environment. The fluorescence of the extrinsic probe, 8-anilino-1-naphthalene-sulfonic acid (ANS), increased only slightly between 5 and 25 °C, and then more sharply between 25 and 40 °C, suggesting a change in conformation leading to a change in either the dissociation constant, K_d, or the number of ANS binding sites, N. The presence of Ca²⁺ ions did not significantly alter the pattern of changes of intrinsic and extrinsic fluorescence with changing temperature. For ANS binding, values of K_d and N were calculated by two different procedures, each based upon different assumptions. The results point to increased exposure of hydrophobic surfaces with increased temperature, strongly supportive of conformational changes. Although more opportunity for hydrophobic interaction leads to increased protein-protein association, turbidity and light-scattering also suggest ion bridge formation between protein molecules. A comparison of the primary sequences of β-caseins from six species reveal residues that are common in all species examined and thus are pivotal in protein folding and conformation, intermolecular hydrophobic interactions and ion bridge formation with Ca²⁺ and inorganic phosphate.