Aggregation, Protein
domain indicating the importance of long-
range interactions in directing the cor-
rect folding. Such species have not been
observed with the C-terminal fragment.
Thus, the occurrence of transient multi-
meric species arising from partially folded
intermediates through hydrophobic inter-
actions does not prevent the correct folding
of a monomeric protein.
Irreversible Aggregation
Thermal unfolding of proteins is fre-
quently accompanied by the formation of
aggregates and therefore behaves as an
irreversible process. It occurs at temper-
atures that vary widely according to the
protein, since the temperature of optimum
stability depends on the balance between
hydrogen bonds and hydrophobic interac-
tions. Generally, the products of thermal
denaturation are not completely unfolded
and retain some structured regions. At the
end of the thermal transition, the addition
of a denaturant such as urea or GdnHCl
frequently induces further unfolding.
An apparent irreversibility at a critical
concentration of denaturant has been ob-
served during the refolding of monomeric
as well as oligomeric proteins. It was re-
ported for the Frst time by M.Goldberg
and coworkers for the refolding of
galactosidase, and for tryptophanase. It
was also observed for a two-domain pro-
tein, horse muscle phosphoglycerate ki-
nase by Yon and coworkers. In the latter
study, when the enzyme activity was used
as a conformational probe of the native
structure, an irreversibility was observed
for a critical concentration of denaturant
equal to 0.7 M
0.1 M GdnHCl, a concen-
tration very close to the end of the transi-
tion curve. Such irreversibility was found
to be concentration dependent. ±or protein
concentrations higher than 30
M, restora-
tion of enzyme activity was practically null.
The formation of irreversible nonnative
species was found to be temperature de-
pendent; it was practically abolished at
C, suggesting that aggregation occurs
through hydrophobic interactions. The ag-
gregation also depends on the time of
exposure of the protein to the denaturant.
When the unfolding–refolding process
was observed using structural signals such
as fluorescence or circular dichroism, it ap-
peared completely reversible whatever the
Fnal denaturant concentration.
Another example is provided by rho-
danese, a two-domain monomeric protein.
During refolding at low denaturant con-
centration, an intermediate accumulates
with partially structured domains and apo-
lar surfaces exposed to the solvent, leading
to the formation of aggregates. The aggre-
gation can be prevented by refolding the
protein in the presence of lauryl maltoside.
Most of the examples discussed above
are related to multidomain proteins. An-
other degree of complexity appears in the
folding of oligomeric proteins. It is gen-
erally accepted that the early steps of the
process are practically identical to the fold-
ing of monomeric proteins. In the last step,
subunit association and subsequent con-
formational readjustments yield the native
and functional oligomeric protein. The cor-
rect recognition of subunit interfaces is
required to achieve the process. The overall
process of the folding of oligomeric pro-
teins was extensively studied by Jaenicke
and his coworkers for several enzymes and
described in reviews. As with monomeric
proteins, the formation of aggregates is
concentration dependent. The kinetics of
aggregation are complex and multiphasic,
indicating that several rate-limiting reac-
tions are involved in the process. In an
attempt to characterize these aggregates, it
was shown that noncovalent interactions
occur between monomeric species with
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