Institute of Physiological Chemistry and Pathobiochemistry, Gutenberg University Mainz, Germany
The Bowman-Birk
inhibitor (BBI) from the soybean is a bifunctional inhibitor of trypsin and
chymotrypsin/elastase characterized by a highly conserved array of 7 disulfide
bonds within a relatively short polypeptide-chain of only 71 amino acids. The
unusually high content of 20% of cysteines in the polypeptide chain of BBI
suggests that this protein may also act as a biological storage of sulfur. The
structure of BBI is characterized by a binary arrangement of an amino-terminal
trypsin-reactive subdomain and a respective chymotrypsin-reactive subdomain. We
have used the double-headed arrangement in the structure of BBI for assessing
the functional and conformational integrity of the variants by comparative
titration and activity determination experiments with trypsin. The
trypsin-reactive subdomain of BBI is stabilized by 4 and the
chymotrypsin-reactive subdomain by 3 S-S bonds. Interdomain disulfide linkages
are not present in this molecule.
The structure
of BBI displays structural peculiarities as exposed hydrophobic patches and
buried ion pairs solvated by internal water molecules in the interdomain
boundary. These features are in marked contrast to most other proteins
comprising a hydrophobic core and exposed polar amino acids. The exposed
hydrophobic patches are a structural peculiarity that is reminiscent of the
kind of structural features that have been postulated to occur in partially
folded proteins. We have hypothesized, that a hydrophobic collapse of the
exposed hydrophobic patches into a regular hydrophobic core could be prevented
by the array of seven disulfide bridges.
We have
assessed the consequences of mutations in the conserved framework of S-S bonds
in the trypsin-reactive subdomain for the autonomous refolding competence and
the enzyme-inhibitory activity of the other subdomain directed against
chymotrypsin. The deletion of disulfide bonds induces dramatic effects on the
refolding competence not only of the trypsin-reactive subdomain, but also on
the activity of the chymotrypsin-reactive subdomain. The variants may be distinguished
into two categories. The first one displays only local irregularities as
expected by hierarchical models of protein folding. In the mutants belong to
the second category the chymotrypsin-inhibitory subdomain is also affected
significantly in its refolding competence and its activity as a result of the
mutations in the trypsin-inhibitory subdomain. Since the mutants belonging to
the second category are located near ionic residues interacting across the
interdomain boundary with ionic residues with opposite charge we have concluded
that ionic interactions may be crucial for the kinetically controlled process
of protein folding in addition to the hydrophobic effect.