Ethanol Controls the Self-Assembly and Mesoscopic Properties of Human Insulin Amyloid Spherulites

Abstract

Protein self-assembly into amyloid fibrils or highly hierarchical superstructures is closely linked to neurodegenerative pathologies as Alzheimer's and Parkinson's diseases. Moreover, protein assemblies also emerged as building blocks for bioinspired nanostructured materials. In both the above mentioned fields, the main challenge is to control the growth and properties of the final protein structure. This relies on a more fundamental understanding of how interactions between proteins can determine structures and functions of biomolecular aggregates. Here, we identify a striking effect of the hydration of the single human insulin molecule and solvent properties in controlling hydrophobicity/hydrophilicity, structures, and morphologies of a superstructure named spherulite, observed in connection to Alzheimer's disease. Depending on the presence of ethanol, such structures can incorporate fluorescent molecules with different physicochemical features and span a range of mechanical properties and morphologies. A theoretical model providing a thorough comprehension of the experimental data is developed, highlighting a direct connection between the intimate physical protein-protein interactions, the growth, and the properties of the self-assembled superstructures. Our findings indicate structural variability as a general property for amyloid-like aggregates and not limited to fibrils. This knowledge is pivotal not only for developing effective strategies against pathological amyloids but also for providing a platform to design highly tunable biomaterials, alternative to elongated protein fibrils.

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

“…Notwithstanding this, spherulites are rarely described. This may be due to the fact that the kinetics of spherulite formation are indistinguashable from the one for fibrils 35,38,55,59 . Moreover, due to their micrometric size, they readily sediment and their presence may be hindered if the sample is not correcly handled.…”

“…To make more intricate this already complex picture, other amyloid-like species (named superstructures) may occur, exhibiting a significantly different -sheet packing when compared to fibrils [30][31][32][33] . Among others, amyloid-like spherical aggregates, named spherulites, ranging from few micrometers to millimeters in diameter can be formed both in vivo and in vitro 34,35 . This aggregate is characterized by a fascinating core-shell morphology and seems to be the result of a self-assembly pattern, which is common for proteins, synthetic polymers and minerals 33,36,37 .…”

“…They are detected in insulin drug formulations 26 and have tunable properties 41 . Regarding the last point, we have recently shown the possibility to form insulin spherulites with distinct morphologies, different molecular structures and physical properties by controlled selfassembly 35 . Interestingly, order-disorder transitions in the formation of protein spherulites may modulate the bio-crystallization of mineral structure 40 .…”

“…Insulin aggregation pathway is indeed considered among the most variegated in vitro aggregation reactions. Depending on the physico-chemical conditions of the formulation, insulin may self-assemble into different structures, from the nm to the mm range and with protein molecules spanning different conformational states 35,39,55 . These parameters may critically determine the immunogenic response induced by the insulin drug product 26 .…”

Zinc Determines Dynamical Properties and Aggregation Kinetics of Human Insulin

“…Insulin, a 5.7 kDa hormone consisting of two mainly alpha-helical chains (A and B) linked by disulfide bonds, can form amyloid fibrils (11,12) and spherulites (13) . The two types of aggregate coexist at low pH when human insulin samples are thermally destabilized (14,15) .…”