Stability matters, too - the thermodynamics of amyloid fibril formation

Abstract

Amyloid fibrils are supramolecular homopolymers of proteins that play important roles in biological functions and disease. These objects have received an exponential increase in attention during the last few decades, due to their role in the aetiology of a range of severe disorders, most notably some of a neurodegenerative nature. While an overwhelming number of experimental studies exist that investigate how, and how fast, amyloid fibrils form and how their formation can be inhibited, a much more limited body of experimental work attempts to answer the question as to why these types of structures form (i.e. the thermodynamic driving force) and how stable they actually are. In this review, I attempt to give an overview of the types of experiments that have been performed to-date to answer these questions, and to summarise our current understanding of amyloid thermodynamics.

Fig. 1. Amyloid thermodynamics. (a) The position of the equilibrium between the soluble and fibrillar state depends strongly on the total protein concentration. (b) Amyloid fibril equilibria need to be defined with respect to I: free monomer concentration, II: fibril length distribution and III: populations of different fibrillar polymorphs (illustrated in different colors).

Fig. 2. Free monomer concentration as a measure of amyloid fibril stability. (a) Depending on the degree of cooperativity of a self-assembling system, a constant monomer concentration as a function of total concentration is reached very abruptly (dotted line, highly cooperative, micellar systems) or more gradually (solid line, e.g. linear polymers, such as amyloid fibrils). (b) In real amyloid systems (here Aβ (1–40)), the free monomer concentration can remain almost indefinitely in a metastable state due to high nucleation barriers preventing the formation of fibrils at concentrations right above c_crit. Reproduced from ref. with permission from Elsevier, copyright 2021. (c) If the solubilities of systematically designed point mutations of an amyloid peptide (Aβ (1–40)) are measured, residue-specific information can be obtained about the importance of different sequence regions for the stability of the fibrillar fold. Reproduced from ref. with permission from Elsevier, copyright 2006. (d) In some scenarios (here α-synuclein in the presence of DMPS liposomes), kinetically trapped states can be reached at which the concentration of amyloid fibrils does not depend on the initially added monomer concentration, but is limited by the lipid concentration (bottom, the red line indicates the concentration of fibrils that would be expected if all the initially added monomer was converted). In this case the free monomer concentration at the plateau of the kinetic experiment followed by thioflavin-T fluorescence (top) is not a measure of the stability of the amyloid fibrils. Reproduced from ref. with permission from Springer Nature, copyright 2015.

Fig. 3. Chemical depolymerisation of amyloid fibrils. (a) One of the earliest such data sets, in which β2-microglobulin amyloid fibrils have been destabilised by GndHCl and the depolymerisation has been followed by CD spectroscopy and ThT fluorescence. Reproduced from ref. with permission from Elsevier, copyright 2004. (b) Global fit of chemical depolymerisation (by urea) of glucagon amyloid fibrils, followed by intrinsic fluorescence. It can be seen that a better fit is achieved with the cooperative linear polymerisation model (solid line) than with the simpler isodesmic linear polymerisation model (dotted line). (c) The dependence of the soluble concentration on the total concentration of glucagon amyloid fibrils is measured at 3 M urea (compare with panel b). In this type of experiment, the isodesmic model does not describe the data. (b) and (c) Reproduced from ref. with permission from the RSC, copyright 2019. (d) Chemical depolymerisation analysis of different amyloid peptides and proteins revealed that the per-residue stability of amyloid fibrils is the highest for short sequences. Reproduced from ref. with permission from the ACS, copyright 2019.

Fig. 4. Non-equilibrium experiments of growth and dissociation to assess amyloid fibril stability. (a) The monomer concentration at which amyloid fibrils are observed neither to grow nor to dissociate corresponds to their equilibrium solubility, c_eq. (b) The growth and dissociation rates of Aβ (1–40) amyloid fibrils attached to an SPR sensor surface were measured at different pH values. It was found that the growth rate, and ultimately the thermodynamic stability of the fibrils greatly increased as the pH was lowered. Reproduced from ref. with permission from the ACS, copyright 2014. (c) Differential scanning fluorimetry experiments to probe amyloid fibril growth and dissociation rates. A temperature variation scheme is shown that includes a brief period at 75 °C, followed by equilibration at 40 °C (lower temperature) and a rapid change to 65 °C (upper temperature), where the dissociation is monitored. Fluorescence emission measured at 350 nm is shown for fibril (F) and monomeric reference (M) samples. The interrupted line indicates the measurement of the initial slope. If the upper temperature is systematically varied, the dissociation rates can be determined at these different temperatures. Reproduced from ref. with permission from Elsevier, copyright 2021.

Fig. 5. The enthalpic signatures of amyloid fibril formation and dissociation. (a) Differential scanning calorimetry (DSC) experiments of β2-microglobulin amyloid fibrils at different NaCl concentrations, from lowest (1) to highest (7). The data show a strong dependence of amyloid thermal stability on salt concentration, as well as a strongly endothermic signature upon dissociation. Reproduced from ref. with permission from Elsevier, copyright 2005. (b) Raw data of isothermal titration calorimetry (ITC) of β2-microglobulin amyloid fibril growth. Small portions of monomer solution are repeatedly injected into a suspension of fibrils. (c) If experiments such as the one shown in (b) are performed at different temperatures, the enthalpies of fibril growth can be determined at these different temperatures, and hence also the heat capacity ΔC_p of the elongation reaction. (b) and (c) Reproduced from ref. with permission from the ASBMB, copyright 2004. (d) When the heat capacities of the elongation reaction of several different amyloid systems are quantified, it is found that they are negative in all cases, i.e. that the enthalpy of the reaction becomes more negative at higher temperatures. Further analysis shows that the magnitude of the heat capacity correlates with the buried hydrophobic surface area upon fibril growth. Reproduced from ref. with permission from the PLoS, copyright 2020.

Fig. 6. Reversal of amyloid fibril formation. (a) Passive compounds. Binding to monomers (left, red compounds) stabilises the soluble state and shifts the equilibrium towards the monomer, leading to fibril dissociation. Binding to fibrils (right, green compound) stabilises the fibrils and shifts the equilibrium towards the fibrillar state. (b) Active compounds are able to destabilise fibrils despite their affinity for the fibrillar state, by undergoing a spontaneous reaction, such as ATP hydrolysis that is coupled with the fibril dissociation reaction. (c) Example of an active compound, a chaperone, dissociating α-synuclein amyloid fibrils. This reaction requires energy in the form of ATP and various co-factors. Reproduced from ref. with permission from CellPress, copyright 2015.

Alexander K. Buell