title: Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains.
abstract: The partly folded states of alpha-lactalbumin (alpha-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 alpha-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 alpha-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 alpha-LA encompasses 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 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 alpha-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 alpha-helix of the corresponding chain segment in native alpha-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 alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-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 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 experiments do correlate with those derived from previous NMR and other physicochemical measurements.
authors.affiliation: CRIBI Biotechnology Centre, University of Padua, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Polverino de Laureto
authors.forename: E
authors.initials: E
authors.lastname: Scaramella
authors.forename: M
authors.initials: M
authors.lastname: Frigo
authors.forename: F G
authors.initials: FG
authors.lastname: Wondrich
authors.forename: V
authors.initials: V
authors.lastname: De Filippis
authors.forename: M
authors.initials: M
authors.lastname: Zambonin
authors.forename: A
authors.initials: A
authors.lastname: Fontana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019286:Endopeptidase K / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D010446:Peptide Fragments; D007768:Lactalbumin; D002918:Chymotrypsin; D019286:Endopeptidase K; D010434:Pepsin A