Limited proteolysis of bovine alpha-lactalbumin: Isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid so lution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of t he single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to b e molten globules and thus protein-folding intermediates. Pepsin was used f or proteolysis at acid pH, while proteinase K and chymotrypsin at neutral p H. The expectations were that these proteolytic probes would detect sites a nd/or chain regions in the partly folded states of alpha-LA sufficiently dy namic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of th e proteolytic events revealed that the fast, initial proteolytic cuts of th e 123-residue chain of alpha-LA in its A-state or apo form by the three pro teases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encom passes the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have bee n isolated by reverse-phase high-performance Liquid chromatography, and the ir conformational properties examined by circular dichroism and fluorescenc e emission spectroscopy. The single chain fragment 53-103, containing all t he binding sites for calcium in native alpha-LA and cross-linked by two dis ulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some stru cture 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 t o 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 confo rmation. Of interest, the proteolytic fragment species herewith isolated co rrespond to the structural domains and subdomains of alpha-LA that can be i dentified by computational analysis of the three-dimensional structure of n ative alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The f ast, initial cleavages at the level of the beta-sheet region of native alph a-LA indicate that this region is highly mobile or even unfolded in the alp ha-LA molten globule (s), while the rest of the protein chain maintains suf ficient structure and rigidity to prevent extensive proteolysis. The subseq uent cleavages at chain segment 95-105 indicate that also this region is so mewhat mobile in the A-state or apo form of the protein. It is concluded th at the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experim ents do correlate with those derived from previous NMR and other physicoche mical measurements.