Microcalorimetry: A Powerful Tool for Characterizing Amyloid Fibril Formation

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 determined^18–21 (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 transitions^20,22,23 (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. K_d 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 K_d 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 K_d 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 K_d. However, single replacement of one of the essential amino acids in the motif (Asp⁴⁹⁶, Leu⁴⁹⁹, or Leu⁵⁰⁰) resulted in binding weaker than 1 mM.