A Free Energy Barrier Caused by the Refolding of an Oligomeric Intermediate Controls the Lag Time of Amyloid Formation by hIAPP

Abstract

Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes. A structural rearrangement includes the F23G24A25I26L27 region of hIAPP, which starts from a random coil structure, evolves into ordered β-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 and 250 μM, which is the thermodynamic parameter that sets the free energy of the oligomers. A 3-state kinetic model fits the experimental data, but only if it includes a concentration independent free energy barrier >3 kcal/mol that represents the free energy cost of refolding the oligomeric intermediate into the structure of the amyloid fibril; i.e., “oligomer activation” is required. The barrier creates a transition state in the free energy landscape that slows fibril formation and creates a stable population of oligomers during the lag phase, even at concentrations below the supercritical concentration. Largely missing in current kinetic models is a link between structure and kinetics. Our experiments and modeling provide evidence that protein structural rearrangements during aggregation impact the populations and kinetics of toxic oligomeric species.

Time dependence of NMR observables reveals salient differences in the accumulation of early aggregated species between human islet amyloid polypeptide and amyloid-β

“…This result is in agreement with recent 2D IR based studies which estimated the supercritical concentration of IAPP to be around 150-250 mM. 36 So neither the micelle reservoir nor a supercritical regime are consistent with our NMR observations. Therefore the very low concentration dependence can be ascribed to saturable microscopic processes.…”

“…Recently, the supercritical concentration of IAPP was estimated to be in the range 150-250 mM, based on 2D IR studies. 36 This indicates that our studied concentration range falls below the supercritical concentration and that the weak concentration dependence cannot be accounted for by a supercritical regime. Therefore such weak concentration dependence implies a saturation of microscopic processes at high monomer concentration, a saturable fibril elongation being most likely.…”

“…31 One of the technique that could detect small oligomeric species (dimer to hexamers) is IMS-MS 34 but it is difficult to estimate the population of such species and evaluate their on-pathway or off-pathway relationships. Finally, the observation of b-sheet oligomers by 2D infrared spectroscopy 35,36 was only possible above the supercritical concentration, requiring highly concentrated samples (about 0.5 mM).…”

“…35 This intermediate was predicted to contain at least 5 monomers and has a parallel b-sheet organisation that differs from the structure of final fibrils, requiring a structural rearrangement during polymerization. 36 In this paper, we wanted to dissect the molecular events of early IAPP aggregation. For that purpose we have conducted a comprehensive set of biophysical studies on IAPP, based on fluorescence, circular dichroism (CD), transmission electron microscopy (TEM), liquid-state NMR and gel electrophoresis.…”

Site-specific detection of protein secondary structure using 2D IR dihedral indexing: a proposed assembly mechanism of oligomeric hIAPP

“…† At 5 min, the peptide is disaggregated and only exhibits the 1650 cm –1 peaks (peak A). At this early time, when the signal is coming from the monomeric species, 58 the isotope label absorbs at about 1595 cm –1 (peak C), is broad 22 cm –1 along the diagonal and weak. Hereafter, we call this the “monomer” spectrum.…”