48
Aggregation, Protein
protoflaments, and then Forms amyloid
fbrils. This Formation is at its maxi-
mum at pH 4.4. Using deuterium–proton
exchange monitored by two-dimensional
NMR spectroscopy on transthyretin at pH
5.75 and 4.5, Liu et al. have shown a se-
lective destabilization oF one halF oF the
β
-sandwich structure oF the protein, in-
creasing the mobility oF this region. These
studies have identifed the residues that
undergo increased conFormational fluc-
tuations under amyloidogenic conditions.
The mutations in the pathological variants
responsible For Familial amyloid polyneu-
ropathies are localized in this region. A
strategy to delay the Formation oF amyloid
fbrils proposed by Saccheti & Kelly was
to develop molecules capable oF stabilizing
the tetramer.
Fig. 11
Molecular model of an amyloid
Fbril derived from cryoelectron
microscopy analysis of Fbrils grown
from an SH3 domain by incubation of a
solution containing the protein at low
pH (reproduced from Dobson, C. (1999)
TIBS
24
, 331, with permission).
Two
variants
oF
human
lysozyme,
Ile56Thr and Asp67His have been reported
to be amyloidogenic; they are responsible
For Fatal amyloidoses. Pepys and colleagues
have determined the precise structures and
properties oF these mutants. The native
Fold oF the two amyloidogenic variants,
as resolved by X-ray crystallography, is
similar to that oF the wild-type protein.
Both variants are enzymatically active, but
have been shown to be unstable. The re-
placement oF an aspartate by a histidine
suppresses a hydrogen bond Formed in
the wild-type protein with a tyrosine in a
neighboring
β
-strand. This rupture opens
a large gap between two
β
-strands. In
the other variant, the replacement oF an
isoleucine by a threonine suppresses a van
der Waals contact with a neighboring he-
lix. Consequently, changes in the interFace
between the
α
-and
β
-domains occur in
both variants, destabilizing the molecule.
The mutations leading to amyloid fbril
Formation are observed to result in a
decreased stability oF the native state. In
all cases, the Formation oF fbrils occurs
From a partially structured molecule via
nucleation-dependent oligomerization. It
was observed For several proteins that
fbrillation takes place only aFter a lag
phase, which is abolished upon seeding.
Nucleation is Followed by the Formation
oF protofbrils whose characteristics have
been determined (±ig. 10). Atomic Force
microscopy and fluorescence correlation
spectroscopy have been used to monitor
transitions
among
the
diFFerent
types
oF assemblies.
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