The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.

In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.

Thermodynamic characterization of the partially unfolded state of Ca(2+)-loaded bovine alpha-lactalbumin: evidence that partial unfolding can precede Ca2+ release.

The thermal denaturation of bovine alpha-lactalbumin (BLA) was studied at pH 7.5 and at various Ca2+ concentrations using near-UV circular dichroism and differential scanning calorimetry. The Ca2+ dependence of the denaturation equilibria proves that, in the transition region, partially unfolded alpha-lactalbumin consists of a mixture of Ca(2+)-loaded and Ca(2+)-free protein. The thermodynamic parameters of the unfolding of these two species were determined at 68 degrees C and were then compared with one other, with the thermodynamic parameters deduced from calorimetric titration of alpha-lactalbumin with Ca2+, and with those derived from Ca2+ titration of a mutant human lysozyme having an engineered Ca(2+)-binding site. This comparison indicated that (a) the unfolding curves for Ca(2+)-BLA deduced from the near-UV ellipticity change are more able to distinguish between unfolding with and without Ca2+ release than those deduced from differential scanning calorimetry, (b) the Ca(2+)-loaded denaturated state of BLA is more folded than the Ca(2+)-free protein at 68 degrees C, and (c) a heat-induced unfolding process, consisting of an initial Ca2+ release, followed by a conformational relaxation, is unlikely to occur at the experimental pH and in the millimolar region of Ca2+ concentrations, due to the large free energy requirement of the initial step. A more probable mechanism would be unfolding via a Ca(2+)-loaded intermediately unfolded state, with subsequent Ca2+ release.

Thermal unfolding of bovine alpha-lactalbumin. Comparison of circular dichroism with hydrophobicity measurements.

The thermal unfolding of apo- and Ca(2+)-loaded bovine alpha-lactalbumin (BLA) determined by circular dichroism measurements at 220 and 270 nm is related to the exposure of its hydrophobic surface measured by the interaction with 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonate. The results indicate that several (about 5) probe molecules are able to bind with low affinity to the compact surface of the native protein. By thermal destabilization to the molten globule state, access is given to two domains with strong hydrophobic character. On further heating the hydrophobic domains degenerate; however, the temperature of destabilization is different for the two domains. A comparable evolution of the hydrophobic behavior is observed for apo- and Ca(2+)-BLA, but the destabilization steps of Ca(2+)-BLA occur at higher temperatures than those of apo-BLA. Also, it has not been reported earlier that, at a moderate Ca2+ concentration (2 mM), Ca2+ remains associated with BLA after the thermally induced destabilization of its native tertiary structure. At 70 degrees C, a partially unfolded state of Ca(2+)-loaded BLA is obtained.