The 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.