Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

Abstract

Ensembles of protein aggregates are characterized by a nano-and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance as biopharmaceutical and its involvement in insulin-derived amyloidosis. By combining Fourier Transform Infrared Microscopy (micro-FTIR) and fluorescence lifetime imaging microscopy (FLIM) we show the occurrence, within the same ensemble of insulin protein aggregates, of a variable -structure architecture and content not only dependent on the species analyzed (spherulites or fibrils), but also on the position within a single spherulite at submicron scale. We unambiguously reveal that the surface of the spherulites are characterized by -structures with an enhanced H-bond coupling compared to the core. This information, inaccessible via bulk methods, allows us to relate the aggregate structure at molecular level to the overall morphology of the aggregates. Our findings robustly solve the problem of probing the ensemble and single particle heterogeneity of amyloid samples. Furthermore, they offer a unique, scalable and ready-to-use screening methodology for in-depth characterization of self-assembled structures, being this translatable to material sciences, drug quality control and clinical imaging of amyloid-affected tissues.

Polysorbate 80 Controls Morphology, Structure and Stability of Human Insulin Amyloid-Like Spherulites

“…Interestingly, double exponential decays were previously observed for ThT in different systems and related to peculiar properties of ThT environment with different rigidity (Stsiapura et al 2008, Thompson et al 2015 or to specific properties of the binding sites (Biancalana and Koide 2010, Sulatskaya et al 2010, Lindberg et al 2015, Ivancic et al 2016, Sidhu et al 2018. In a previous study using phasor approach for insulin spherulites samples, we found ThT fluorescence decays with same characteristic lifetimes as shown in the present study (De Luca et al 2020). We attribute ThT lifetime changes within different aggregates structures to the superimposition of two main effects:…”

“…the rigidity of the environment (due to the molecular-rotor nature of ThT (Stsiapura et al 2008, Thompson et al 2015 and the specificity of ThT binding site, which depends on the details of the βsheet architecture. More specifically, we established a clear correlation between differences in ThT lifetime and peculiarities of intermolecular β-structures: higher ThT lifetimes correspond to higher content of β-structures (De Luca et al 2020). Based on that, data in Figure 5a and 5b suggest that PS80 induced the formation of spherulites with higher content of intramolecular -structures.…”

“…In our case we can identify two characteristic ThT lifetimes at 1=0.7 ns (blue circle) and 2=2.4 ns (red circle). Distances between each lifetime distribution and the single exponential phasors on the universal circle (F1 and F2) represent the fraction of each component (Digman et al 2008, De Luca et al 2020). In the insert in Figure 5a, we report the obtained F1 fractions as a function of the PS80 concentration and in Figure 5b representative 256 × 256 lifetime maps obtained for samples at different PS80 concentrations were color coded on a scale from blue (pure fast component, 1 =0.7 ns) to red (pure slow component, 2=2.4 ns).…”

“…For both samples, the spectra were normalized to 1675 cm -1 and characterised by two main broad components centred at 1620 cm -1 and 1670 cm -1 . The 1620 cm -1 peak is assigned to the aggregated -sheets characteristic of the amyloid structure, while the peak at 1670 cm -1 represents the presence of -helices, turns and loops (1660 cm -1 ) and antiparallel aggregated -sheets (1670-1680 cm -1 ) (De Luca et al 2020). Insulin spherulites formed at high concentration of PS80 had a higher content of aggregated -sheets compared to the aggregates formed in absence of PS80, confirming the change in internal structure inferred from the FLIM analysis.…”

Spherulite‐Like Micelles

“…Amyloid spherulites have been detected in vivo for the Alzheimer peptide Aβ and in mice expressing mutations of the human prion protein [9, 10] . The search for effective prevention and treatment strategies has led to substantial research efforts into understanding the properties and structures of these protein aggregates [5–9, 11] …”