Many soluble proteins misfold and form insoluble
amyloid fibrils. This process occurs in many neurodegenerative
disorders, such as Alzheimer’s disease,
amyotrophic lateral sclerosis, Parkinson’s disease,
and Huntington’s disease.1–5 Amyloid
formation has also been implicated in transmissible
spongiform encephalopathies (prion diseases) such
as Creutzfeldt-Jakob disease and hereditary familial
amyloid diseases.6
The structure and morphology of amyloid proteins
have been studied by atomic force microscopy, nuclear magnetic resonance, electron microscopy, X-ray diffraction,
and spectroscopic methods.4,7–9 Amyloid fibrils
have a cross β structure whereby β strands are perpendicular
to the axis of the fibrils.1 More than
proteins and peptides are known to form amyloids in
vitro under appropriate conditions of temperature,
pH, and/or pressure.
The current model for prion diseases is that the
infectious agents (prions) are protein. Prions promote
the conversion of host protein to an insoluble
diseased form.10 There are conformational changes
between the native and disease states of prion proteins,
since no covalent modifications differentiate
the two proteins.
There have been several structural studies
of amyloid fibrils, including different
forms of prion proteins.11,12 There is a
relationship between the thermodynamic
and conformational stability of
soluble proteins and their ability to form
amyloid fibrils.13–16 This research is used
to develop models for amyloidogenesis
and prionogenesis.
Research has been conducted on ligands
which stabilize the native soluble protein,
thereby reducing the formation of amyloid
fibrils,17 as well as characterization of protein–protein interactions involved in proteolysis
and prion formation. This research
may lead to the development of therapeutic
agents in the treatment and prevention of
amyloid-related diseases.
This application note discusses the use of differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and pressure
perturbation calorimetry (PPC) in the study of amyloid
formation and prion proteins. The reader may refer to
cited articles for detailed experimental design, results,
and discussion.
Overview of microcalorimetry
Isothermal titration calorimetry is a technique
for monitoring any chemical reaction
initiated by the addition of a binding partner,
and is the method of choice for characterizing
biomolecular interactions. When
two components bind, heat is either generated
or absorbed. Measurement of this heat
allows accurate determination of binding
constants (KB), reaction stoichiometry (n),
enthalpy (ΔH), and entropy (ΔS). In a single
experiment, a complete thermodynamic
profile of the molecular interaction can be
determined18–21 (Figure 1).
Figure 1 - Typical ITC data.
Differential scanning calorimetry measures
the heat changes associated with thermal
denaturation of a biomolecule. DSC is
used for protein unfolding and stability
studies and the thermodynamics associated
with these transitions20,22,23 (Figure 2).
Figure 2 - Typical DSC data.
Pressure perturbation calorimetry measures
the heat change resulting from a
pressure change above the protein solution.
PPC determines changes in volume
due to protein unfolding and calculates
coefficients of thermal expansion,
thereby determining the volumetric properties
of proteins.24–26
Microcalorimetry uses native materials,
and there is no need for labeling, chemical
modification, or immobilization. In
addition, calorimetry is the only method
that directly measures the heat change associated with biomolecular interactions, directly
resulting in thermodynamic parameters.
Thermodynamics of Congo Red binding to amyloid proteins
Congo Red (CR) is a symmetrical sulfonated azodye
consisting of a hydrophobic center. The binding of
CR to proteins has been reported to inhibit or
enhance amyloid fibril formation; CR binding is
used as a diagnostic stain for amyloid fibrils. CR has
been reported to stabilize αβ monomer and inhibit
its oligomerization to inhibit conversion of normal
prion protein to its aggregation-prone pathogenic
form, and reduce αβ amyloid neurotoxicity. CR and
its analogs have also been studied as potential therapeutic
agents to inhibit amyloid fibril formation.
High CR concentrations inhibited amyloid fibril formation,
although at low CR concentrations amyloid
formation was enhanced.
Kim et al.27 used ITC to study binding thermodynamics
of CR to VL SMA, an amyloidogenic immunoglobulin
light chain variable domain. Stoichiometry of
binding was approx. 3.4 mol of CR per mol of SMA at
25 °C to approx. 5 at 30 °C. The authors proposed that
increased binding was due to exposure to more binding
sites as temperature increased. Kd values were in the
μM range; these results correlated to values determined
by other methods.
The structure of CR suggested that binding could be
through hydrophobic interactions, electrostatic interactions,
or a combination of both. ITC experiments with
different CR/SMA ratios, at different temperatures,
showed that CR binding to the protein was enthalpically
driven, a characteristic of binding primarily by hydrogen
bonding and electrostatic interactions (Figure 3).
Thermodynamic data from ITC suggested that
hydrophobic interactions did not occur. The authors proposed
that binding was via the interaction between the sulfate groups of the CR and the positively charged
amino acids of the protein. Previous studies indicated
that binding of CR to other proteins susceptible to amyloid
formation was also due to electrostatic interactions.
Figure
3 - Thermodynamic parameters for interactions between recombinant
SMA and Congo Red, determined by ITC (data from Ref. 27). A: 20.1 μM
SMA dimer in ITC cell, 760 μM Congo Red in ITC syringe, 25 °C. B: 32.3
μM SMA dimer in ITC cell, 1036 μM Congo Red in ITC syringe, 25 °C. C:
20.1 μM SMA dimer in ITC cell, 760 μM Congo Red in ITC syringe, 30 °C.
Experimental methods described in Ref. 27.
Binding of retinol binding protein–Vitamin A complex to transthyretin
Patients with transthyretin (TTR)-associated amyloid
diseases, familial amyloid neuropathy (FAP), and senile
systemic amyloidoses have amyloid fibrils accumulated
in tissue. TTR is a tetrameric protein found in plasma
and cerebral spinal fluid. The mechanism of amyloid
fibril formation from TTR involves tetramer dissociation
to monomer intermediate, and under acidic conditions,
the monomer forms amyloid fibrils. Thyroxin
(T4 thyroid hormone) binds to TTR tetramer and stabilizes
the protein, inhibiting amyloid formation.
White and Kelly28 used ITC to study the binding stoichiometry
of TTR and retinol binding protein–Vitamin
A complex (holoRBP). Retinol binding protein (RBP)
circulates in plasma and binds Vitamin A (retinol) and
tetrameric TTR. This interaction prevents glomerular filtration
of RBP by the kidneys. ITC experiments at neutral
pH showed that approx. 2 mol of recombinant
holoRBP bound per mol of TTR, and Kd was approx. 300
nM. These correlated with values determined by other
experimental methods of holoRBP isolated from plasma.
ITC experiments could not be performed at low pH
because TTR fibril formation was rapid at the concentration
used for ITC (50 μM). The authors used analytical centrifugation and estimated that Kd was lower
than 7.2 μM in the pH range of 4.4–7.6. This demonstrated
that the binding interaction between TTR and
holoRBP was retained under acidic conditions, allowing
for a mechanism for holoRBP affecting TTR amyloid
fibril formation. Fibril formation studies confirmed
that holoRBP inhibits TTR fibril formation. ITC
established that the binding stoichiometry of interacting
molecules RBP, Vitamin A, and T4 influenced
TTR amyloidogenicity in vitro. The authors estimated
that at least 70 genes are responsible for controlling
holoRBP and T4 binding stoichiometry to TTR.
Thermodynamics of amyloid formation
Kardos et al.29 investigated the thermodynamics of
amyloid formation as a way to understand the morphology and structure of amyloids. They
used the model system β2-microglobulin
(β2m), a protein responsible for dialysis-related
amyloidosis.
Native β2m in plasma is a monomer, and
its structure has seven β strands organized
into two β sheets connected by a disulfide
bond. When β2m is acid denatured, the
protein forms an ordered, cross β sheet
structure of amyloid fibrils. β2m monomers
are formed in vitro by a seed-dependent
fibril extension, where seeds (fragmented
fibrils) are added to monomer.
ITC was used to measure the enthalpy and
heat capacity changes associated with the
seed-dependent extension of β2m amyloid
fibrils under acidic conditions.29 A single
injection of monomer into seed solution
(or vice versa) started the fibril formation.
ΔH of fibril formation was measured by
ITC, and the kinetics of reaction via
enthalpy production was also determined.
The authors measured heat capacity change (ΔCp)
by performing ITC at different temperatures. The
morphology changed from monomer to fibril, but the
ΔCp of amyloid formation was similar to that of the
folding of native globular protein, suggesting that
amyloid fibrils and globular proteins had a similar
overall surface burial of polar and charged groups.
ΔH of fibril formation was lower than that of globular
proteins, suggesting that amyloid fibrils had a
lower level of internal packing and a lower level of
amino acid side chain packing in the amyloid fibrils.
Thermodynamic data led to the proposal that fibril
formation was an entropy-driven process.
Protein–protein interactions
Proteolysis is a key step in the morphology of amyloidoses,
and knowledge of the interaction of protease
with target protein is important. Memapsin 2
(β-secretase) is a protease that initiates the cleavage
of β-amyloid precursor protein (APP). Memapsin 2
and APP are then transported from the cell surface
to the endosomes. In the endosomes, APP proteolysis
by memapsin 2 and γ-secretase produces amyloid
β. When amyloid β protein accumulates in the
brain, this leads to neuron death and onset of
Alzheimer’s disease. The cytosolic domain of
memapsin 2, containing acid cluster dileucine motif
(sequence DISLL), binds to the VHS (Vps-27, Hrs,
and STAM) domain of GGA (Golgi-localized γ-ear
containing ARF binding) proteins. This binding is
believed to be the recognition step for the vesicular
packaging of memapsin 2 prior to transport into
endosomes. Serine phosphorylation in the binding
domain of memapsin 2 was reported to be the regulatory
step in recycling of memapsin from endosomes
to the cell surface.
He et al.30 used ITC to study the binding of
memapsin 2 cytosolic peptides to wild-type VHS
binding domain of three different GGA proteins
(GGA1, GGA2, and GGA3). Table 1 summarizes
these results. Using different peptide sequences, they
confirmed the importance of DISLL motif for binding
to VHS. The replacement of isoleucine-serine or
the aspartic acid preceding the motif did not significantly
change the Kd. However, single replacement
of one of the essential amino acids in the motif
(Asp496, Leu499, or Leu500) resulted in binding weaker
than 1 mM.