194
j. Y. CHANG
The folding intermediates of hirudin and TAP are actually far more heterogeneous than what has appeared on HPLC as shown in Fig. 13.1 and Fig. 13.2. Take
the 5 min trapped intermediates from the hirudin "control minus" folding as an
example (Fig. 13.1). Each HPLC fraction, when isolated and further analyzed by
capillary electrophoresis, was found to contain a mixture of complex species. If
all these species are added up, it approaches the possible numbers of the disulfide
isomers (there are 60 possible 1- and 2-disulfide isomers for a protein containing
3 disulfides). This high heterogeneity was similarly observed in the cases of TAP
(Chang, 1996), PCI (Chang et aI., 1994), and EGF (Chang et aI., 1995).
4.2
Redox Agents Promote the Efficiency of Folding,
but do not Alter the Composition of Folding Intermediates
Redox reagents are common ingredients for the oxidative folding of disulfide
containing proteins (Sexena & Wetlaufer, 1970; Creighton, 1986; Lyles & Gilbert,
1991). A systematic study has been performed to examine the effect of GSHI
GSSG and CyslCys-Cys on the folding mechanism of hirudin (Chang., 1994), TAP,
PCI and EGF. These studies include evaluation of single redox agent as well as
combined redox components at varying concentrations. Our results allow us to
reach two important conclusions (Chang., 1994). First, the redox agents accelerate the kinetics and promote the efficiency of folding, but do not alter the composition of folding intermediates. For each of these four proteins, the composition
of folding intermediates, as judged by their HPLC patterns, remains indistinguishable under various combinations of redox agents. Second, redox agents promote the folding of hirudin, TAP, PCI and EGF in a two-stage manner. GSSG and
Cys-Cys accelerate the disulfide formation, whereas GSH and Cys catalyze the
disulfide reshuffling. As a consequence, the kinetic of the flow of folding intermediates and the level of their accumulation along the folding pathway are dependent upon the concentration of redox agents. When the folding of PCI was carried out in the buffer containing GSSG or Cys-Cys, rapid formation of the disulfide bonds leads to the accumulation of 3-disulfide scrambled PCI as the folding
intermediates (Fig. 13.3) (Chang et aI., 1994). For instance, under 2 mM of CysCys, the only detectable folding intermediates after 1 min of folding are scrambled PCl. The inclusion of both Cys-Cys and Cys (or both GSSG and GSH)
resulted in a simultaneous disulfide reshuffling of the scrambled species and the
rapid recovery of the native structure. This two-stage mechanism has been
observed with all four proteins analyzed in our laboratory.
4.3
Nearly all Possible Isomers of Scrambled Species of Hirudin
and TAP were Shown to Exist Along the Folding Pathway
The complexity of the folding intermediates of hirudin and TAP suggests that
nearly all possible disulfide isomers may exist along the folding pathway. Among
the three groups of folding intermediates, however, the 3-disulfide scrambled
species represents the only class of intermediates that can be purified to homoge-
j. Y. CHANG
The folding intermediates of hirudin and TAP are actually far more heterogeneous than what has appeared on HPLC as shown in Fig. 13.1 and Fig. 13.2. Take
the 5 min trapped intermediates from the hirudin "control minus" folding as an
example (Fig. 13.1). Each HPLC fraction, when isolated and further analyzed by
capillary electrophoresis, was found to contain a mixture of complex species. If
all these species are added up, it approaches the possible numbers of the disulfide
isomers (there are 60 possible 1- and 2-disulfide isomers for a protein containing
3 disulfides). This high heterogeneity was similarly observed in the cases of TAP
(Chang, 1996), PCI (Chang et aI., 1994), and EGF (Chang et aI., 1995).
4.2
Redox Agents Promote the Efficiency of Folding,
but do not Alter the Composition of Folding Intermediates
Redox reagents are common ingredients for the oxidative folding of disulfide
containing proteins (Sexena & Wetlaufer, 1970; Creighton, 1986; Lyles & Gilbert,
1991). A systematic study has been performed to examine the effect of GSHI
GSSG and CyslCys-Cys on the folding mechanism of hirudin (Chang., 1994), TAP,
PCI and EGF. These studies include evaluation of single redox agent as well as
combined redox components at varying concentrations. Our results allow us to
reach two important conclusions (Chang., 1994). First, the redox agents accelerate the kinetics and promote the efficiency of folding, but do not alter the composition of folding intermediates. For each of these four proteins, the composition
of folding intermediates, as judged by their HPLC patterns, remains indistinguishable under various combinations of redox agents. Second, redox agents promote the folding of hirudin, TAP, PCI and EGF in a two-stage manner. GSSG and
Cys-Cys accelerate the disulfide formation, whereas GSH and Cys catalyze the
disulfide reshuffling. As a consequence, the kinetic of the flow of folding intermediates and the level of their accumulation along the folding pathway are dependent upon the concentration of redox agents. When the folding of PCI was carried out in the buffer containing GSSG or Cys-Cys, rapid formation of the disulfide bonds leads to the accumulation of 3-disulfide scrambled PCI as the folding
intermediates (Fig. 13.3) (Chang et aI., 1994). For instance, under 2 mM of CysCys, the only detectable folding intermediates after 1 min of folding are scrambled PCl. The inclusion of both Cys-Cys and Cys (or both GSSG and GSH)
resulted in a simultaneous disulfide reshuffling of the scrambled species and the
rapid recovery of the native structure. This two-stage mechanism has been
observed with all four proteins analyzed in our laboratory.
4.3
Nearly all Possible Isomers of Scrambled Species of Hirudin
and TAP were Shown to Exist Along the Folding Pathway
The complexity of the folding intermediates of hirudin and TAP suggests that
nearly all possible disulfide isomers may exist along the folding pathway. Among
the three groups of folding intermediates, however, the 3-disulfide scrambled
species represents the only class of intermediates that can be purified to homoge-