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The pathways of unfolding and refolding of three homologous proteins are shown
to be closely related. This implies that folding pathways, as well as the final
folded conformation, have been largely conserved during the presumed
evolutionary divergence of these proteins from a common ancestor. The pathways
of the homologous proteins I and K from black mamba venom were determined here,
using the disulphide interaction between their six cysteine residues to trap and
identify the intermediate states, and are compared with those determined
previously in the same way for the homologous bovine pancreatic trypsin
inhibitor. The major one- and two-disulphide intermediates are the same with all
three proteins; their kinetic roles are similar, although there are differences
in the rates at which they are interconverted and in the minor intermediates
that accumulate. As a consequence, different pathways may predominate with
another homologous protein, even though the various most favourable pathways are
the same. The energetics of the folding transitions and the stabilities of the
folded states differ substantially for the three proteins. The differences in
stabilities of the fully folded states are primarily reflected kinetically in
the rate-determining rearrangements of the native-like conformation; the rates
and equilibria of the other steps are not affected markedly. With the less
stable proteins, the direct folding pathway of sequential formation of the three
correct disulphide bonds becomes significant and is the most facile when
considered on a solely intramolecular basis.
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