Limited proteolysis of bovine α‐lactalbumin: Isolation and characterization of protein domains
Authors: Laureto Patrizia Polverino De , Scaramella Elena , Frigo Marta , Wondrich Francesca Gefter , Filippis Vincenzo De , Zambonin Marcello , Fontana Angelo 
Published: February 2009

AbstractThe partly folded states of α‐lactalbumin (α‐LA) exposed to acid solution at pH 2.0 (A‐state) or at neutral pH upon EDTA‐mediated removal of the single protein‐bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein‐folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of α‐LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time‐course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123‐residue chain of α‐LA in its A‐state or apo form by the three proteases occur at the same chain region 39‐54, the actual site(s) of cleavage depending upon the protease employed. This region in native α‐LA encompasses the β‐sheets of the protein. Subsequent cleavages occur mostly at chain regions 31‐35 and 95‐105. Four fragment species of α‐LA have been isolated by reverse‐phase high‐performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53‐103, containing all the binding sites for calcium in native α‐LA and cross‐linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of α‐helix of the corresponding chain segment in native α‐LA. Evidence for some structure was also obtained for the two‐chain species 1‐40 and 104‐123, as well as 1‐31 and 105‐123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1‐34 connected to fragment 54‐123 or 57‐123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native‐like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of α‐LA that can be identified by computational analysis of the three‐dimensional structure of native α‐LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872‐884). The fast, initial cleavages at the level of the β‐sheet region of native α‐LA indicate that this region is highly mobile or even unfolded in the α‐LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95‐105 indicate that also this region is somewhat mobile in the A‐state or apo form of the protein. It is concluded that the overall domain topology of native α‐LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of α‐LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.