Research Report 2000 - MDC

Folding and
Misfolding of Proteins
Gregor Damaschun
The creation of proteins in living cells
consists of two main processes:
biosynthesis of the polypeptide chain
and its folding into the native, threedimensional
structure with biological
function. The first process has been
thoroughly studied, while the second
process is less well known. We have
learnt in recent years that the proteinfolding
process is not always flawless
within the cell and this can have
pathological consequences. Thus, a
number of human diseases are related
to the deposition of protein fibrils
causing tissue damage and
degeneration. Amyloid fibrils develop
from abnormal, misfolded
conformational states of different
normally soluble proteins forming
ordered aggregates. The reasons for
misfolding are unknown. Therefore,
there are no causal treatments for
these diseases. The group “Physics of
Biopolymers” is engaged in studies of
the folding pathways of proteins to
understand the causes of misfolding.
The main experimental methods used
in these studies include solution X-ray
scattering (SOXS), dynamic light
scattering (DLS) and optical
spectroscopy, including kinetic
techniques. Methods of statistical
physics of chain molecules have been
applied to modelling the experimental
data.
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Polymorphism of proteins
Textbooks state that the structure of a
protein is determined by its amino
acid sequence. However, we have
been able to show experimentally that
this so-called second genetic code is
not unambiguous. The threedimensional
structure of a protein is
determined not only by the amino acid
sequence but also by the environment
of the protein molecules and is
influenced by interactions between
structural intermediates on the folding
pathway. Therefore, many proteins
can adopt differently folded threedimensional
structures and only one
of these structures is functionally
active. For yeast phosphoglycerate
kinase (PGK), we observed in
addition to the native structure two
further, different conformations. The
starting point for the formation of
these misfolded conformations is the
acid-unfolded state. At low pH values,
PGK has the conformation of an
expanded random walk. If the
molecule is transferred to a
hydrophobic environment with a low
dielectric constant, the entire molecule
forms α-helix. On the other hand,
anion-induced partial refolding of the
acid-unfolded state leads to the
formation of amyloid-like fibrils. Half
the amino acids have the conformation
of a cross-β-helix which is typical of
all amyloids.
Folding pathways and kinetics
The formation of amyloids starts from
non-natively folded monomeric
intermediates. The monomers
aggregate forming successively
dimers, tetramers and octamers. More
and more cross-β-structure develops
during this aggregation process. The
kinetics of aggregation is strongly
dependent on protein concentration.
At room temperature, this process
may take several hours. Subsequently,
the octamers grow in one direction
only and form fibrils. The growth of
the fibrils, i.e. their time-dependent
elongation, may take some months.
Our results indicate that inhibitors of
cross-β-structure formation can be
effective only during the early phases
of amyloidosis. The slow kinetics are
typical of misfolding of proteins into
amyloids. In vivo, the progression of
these processes is in some cases even
slower than in our in vitro experiments.
By contrast, the folding of a protein
into its native structure is a fast
process. Typical times for folding vary
from milliseconds to minutes. One
central problem in protein folding is
the question, whether chain segments
with a periodic secondary structure
develop in a first step, then form in a
second step the compact globule
through diffusion (framework model),
or whether the chain initially
collapses, driven by hydrophobic
interactions, with concurrent or
subsequent formation of segments
with periodic secondary structure
(hydrophobic collapse model). We
have been able to show experimentally
that both models are not general
alternatives. There are proteins folding
mainly according to the mechanism of
the framework model (e.g., bovine
RNase A) as well as folding according
to the hydrophobic collapse model
(e.g., bovine α-lactalbumin). Further
studies are necessary to address the
open question: which of these folding
scenarios is more prone to the
misfoldings that lead to amyloids?
Up to now, a search for common
properties of amyloid-forming
proteins has been unsuccessful.