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.
Vanderheeren
G
G
Interdisciplinary Research Center, Katholieke Universiteit Leuven, Kortrijk, Belgium.
Hanssens
I
I
Meijberg
W
W
Van Aerschot
A
A
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; D007768:Lactalbumin; D009113:Muramidase; D002118:Calcium