Research Report 2000 - MDC

Protein Folding and
Misfolding
Heinz Fabian
Understanding the mechanism of
protein folding is of considerable
clinical importance since a number of
diseases, such as Alzheimer’s disease
and spongiform encephalopathies, are
pathological consequences of
misfolding. A characteristic feature of
various medical disorders is the selfassembly
of β-sheet domains resulting
in the formation of pathogenic protein
aggregates (amyloid fibrils). A
growing body of data suggests that
partially folded intermediates are
precursors of such aggregates. Fourier
transform infrared (FTIR) spectroscopy
is particularly good at detecting the
presence of and changes in β-sheet
structures. Moreover, the FTIR
approach now allows the investigation
of in vitro protein unfolding/folding
events in the millisecond to minute
time scale and we have applied
infrared spectroscopic techniques to
investigate folding and misfolding
processes in peptides and proteins.
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Peptides and proteins capable
of forming amyloid fibrils in vitro
in collaboration with E.-G. Krause
(Research Institute for Molecular
Pharmacology, Berlin)
The principles behind β-sheet
formation are not well understood due
to difficulties in the development of
simple model systems. The design of
β-sheet peptides is complicated by
their limited solubility in water and
due to the nature of their folding,
which is dictated by long range
interactions. We have described de
novo β-sheet peptides which selfassemble
into fibrillar structures. The
influence of peptide length,
concentration, and D-amino acid
substitution on the ability to form
amyloid fibrils has been analysed. Our
results suggest that amyloid formation
is not restricted to very few peptide
sequences associated with disease
states. Conformational studies of
synthetic analogs of Alzheimer βA4
peptides have revealed that the central
hydrophobic region plays a key role in
the conformational switch of the
peptide.
in collaboration with G. Damaschun
(MDC)
Although unrelated to proteins
involved in known amyloid diseases,
phosphoglycerate kinase is capable of
forming amyloid fibrils under certain
conditions. The ability to design
conditions under which fibril formation
can be observed with otherwise
soluble proteins offers the opportunity
to investigate the molecular
mechanism of the underlying process.
In the case of yeast phosphoglycerate
kinase, a rapidly formed and partially
folded monomeric intermediate
involved in the aggregation process
has been detected.
Folding of the enzyme
ribonuclease T1
in collaboration with D. Naumann
(Robert Koch-Institute, Berlin)
Folding of the model protein
ribonuclease T1 is known to be
complex, involving several fast and
slow phases. Our time-resolved
infrared studies have provided new
insights into the structural events
accompanying the folding of
ribonuclease T1. In particular, an
extremly slow folding process has
been observed, which was correlated
with restricted structural changes due
to an isomerization of the proline-39
bond in the protein.
Unfolding and folding of the
lambda Cro repressor protein
in collaboration with V.V. Rogov
(Institute of Protein Research,
Russia), K. Gast (MDC) and
H.H. Mantsch (Institute for
Biodiagnostics, Canada)
The λ-Cro repressor is one of the
proteins which can be used as a model
system to study the interplay between
changes in secondary structure and the
state of association upon unfolding
and refolding. In the active state of the
Cro repressor protein, two monomeric
units form a dimer by aligning the
C-termini of each monomer, allowing
the formation of an antiparallel
β-ribbon across the dimer. The
N-terminal parts form small globular
subdomains that consist of three
α-helices and a short N-terminal
β-strand connected to the β-ribbon.
Conventional FTIR and dynamic light
scattering experiments have shown
that the first thermal transition of a
variant of Cro, which contains a
disulfide cross-link between the
protein subunits in the dimer, only
involves unfolding of the three
α-helices and the short N-terminal
β-strand. The intermediate state has a
well structured intermolecular β-sheet
domain still formed by the C-terminal
parts of each polypeptide chain and
associates into a tetrameric structure.
This stable intermediate unfolds
during a second transition at higher
temperatures, a process that is also
accompanied by dissociation of the
tetramers. Our kinetic infrared studies
have revealed that oligomerization
of the covalently cross-linked protein
strongly decelerates its folding.
Analysis of structural changes
applying 2D-IR correlation
spectroscopy, a novel experimental
approach, has provided fundamental
insights into sequential events in the
formation and also unfolding of the
stable intermediate of the Cro protein.