34
Aggregation, Protein
As can be seen here, several mecha-
nisms exist, which lead to the formation
of aggregates. It is recognized that ag-
gregation results from the association of
incompletely or incorrectly folded interme-
diates through hydrophobic interactions.
In the energy landscape of protein folding,
the presence of local minima separated
by an energy barrier allows the accumu-
lation of intermediates. If the barrier is
high enough, these intermediates cannot
easily reach the native state, and kinetic
competition thus favors the formation
of aggregates.
3
Protein Folding in the Cellular Environment
3.1
Molecular Crowding in the Cells
The main rules of protein folding have
been deduced from a considerable body of
in vitro
and
in silico
studies. It has been
accepted that the same mechanisms are
involved in
in vitro
refolding and in the
folding of a nascent polypeptide chain in
the cell. However, the intracellular envi-
ronment differs markedly from that of
the test tube where low protein concen-
trations are used. The interior of a cell is
highly crowded with macromolecules. The
concentration is so high that a signiFcant
proportion of the volume is occupied. As
mentioned by Ellis, in general, 20 to 30%
volume of the interior of the cells are oc-
cupied by macromolecules; for example,
the concentration of total protein inside
cells ranges from 200 to 300 g L
1
.The
total concentration of proteins and RNA
inside
Escherichia coli
ranges from 300 to
400 g L
1
depending on the growth phase.
Polysaccharides
also
contribute
to
the
crowding. It can be predicted practically
that diffusion coefFcients will be reduced
by factors up to 10-fold due to crowding.
Since the average time for a molecule to
move a certain distance varies by
D
2
,
D
being the diffusion coefFcient, it will
take 100 times longer to move this dis-
t
an
c
einth
ec
e
l
la
swou
ldb
en
e
c
e
s
s
a
r
y
under low concentration conditions. An-
other prediction indicates that equilibrium
constants for macromolecular associations
maybeinc
reasedbytwototh
reeo
rde
rs
of magnitude.
Molecular crowding inside cells also has
consequences for protein folding, favoring
the association of partly folded polypeptide
chains into aggregates. This could explain
why cells contain molecular chaperones,
even
though
most
denatured
proteins
refold spontaneously in the test tube.
3.2
The Role of Molecular Chaperones
The discovery of a ubiquitous class of
proteins mediating the correct folding in
cellular environment has led to a recon-
sideration of the mechanism of protein
folding
in
vivo
.
Historically,
the
term
molecular chaperone
was introduced by
Laskyard and coworkers in 1987 to de-
scribe
the
function
of
nucleoplasmin,
which mediates the
in
vitro
assembly
of nucleosomes from separated histones
and DNA. The concept was further ex-
tended
by
Ellis
to
deFne
a
class
of
proteins
whose
function
is
to
ensure
the
correct
folding
and
assembly
of
proteins through a transient association
with the nascent polypeptide chain. Stud-
ies on heat-shock proteins have widely
contributed to the development of this
concept.
Today, more than 20 protein families
have been identiFed as molecular chap-
erones. Molecular chaperones comprise
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