Molecular medicine-Detection of Aggregates during the Refolding Process

Transient Aggregation
Several observations indicate that transient
aggregation could occur during in vitro
protein refolding. Direct evidence for the
28 Aggregation, Protein
transient association of intermediates has
been obtained from small angle X-ray
scattering, in the case of apomyoglobin
by Doniach and his group, and in the
case of carbonic anhydrase by Semisotnov
and Kuwajima, and by Silow et al.
During the refolding of phosphoglycerate
kinase, rapidly transient multimeric
species (dimers, trimers, and tetramers)
yielding to the native monomeric protein
have been detected by Pecorari et al. These
species are not in equilibrium, but are
formed rapidly and disappear in the slow
folding step. Unlike classical aggregates,
their distribution does not depend on protein
concentration, and they are produced
at concentrations as low as 0.05 μM. The
distribution of the oligomers is completely
established at the end of the fast refolding
step. To take into account all these observations,
a model, which is formally similar
to a reaction of copolymerization between
two types of monomers, has been proposed.
In this model, the refolding of the
protein produces two types of intermediate
conformers that can associate with the
same or the other type. In the latter case,
the association cannot be extended further
(Fig. 2). Transientmultimeric species have
also been observed during the refolding
of the isolated N-terminal domain under
conditions in which neither the whole
native protein nor the folded isolated Ndomain
associate. However, they cannot
transform to the native form in the absence
of the interactionswith the complementary
Denatured protein
Folded
protein
k1 k2
Fast phase Slow phase
Fig. 2 Model proposed for the formation of
transient multimeric species during the refolding
of yeast phosphoglycerate kinase. Two types of
conformers are produced in the early step of
folding. One of these can be directionally
extended by association with either the same
conformer or another type of conformer. In this
last case, the association cannot be further
extended. The distribution of species results
from a kinetic competition between two kinetic
processes. (Reproduced from Pecorari, F.,
Minard, P., Desmadril, M.,Yon, J.M. (1996)
Occurrence of transient multimeric species
during the refolding of a monomeric protein, J.
Biol. Chem. 271, 5270–5276.)
Aggregation, Protein 29
domain indicating the importance of longrange
interactions in directing the correct
folding. Such species have not been
observed with the C-terminal fragment.
Thus, the occurrence of transient multimeric
species arising from partially folded
intermediates through hydrophobic interactions
does not prevent the correct folding
of a monomeric protein.