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


Abstract

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. Study holds ProTherm entries: 4669 Extra Details: transition region; CaCl2-binding site;,conformational relaxation; intermediately unfolded state

Submission Details

ID: iPhhWzCP4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Vanderheeren G;Hanssens I;Meijberg W;Van Aerschot A,Biochemistry (1996) Thermodynamic characterization of the partially unfolded state of Ca(2+)-loaded bovine alpha-lactalbumin: evidence that partial unfolding can precede Ca2+ release. PMID:8988012
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