Standardization of cross-site biophysical studies of bovine insulin amyloids is challenged by structural polymorphism

Protein structure can be analysed using a range of different techniques. Many of the available principal techniques for assessing protein structure are developed and optimized for natively folded proteins that are soluble. Amyloids are an alternative protein fold, often associated with protein malfunction and disease but are also found as the functional fold for some proteins. While functional amyloids, just like soluble globular proteins, commonly attain one specific and precise fold to exert its function, disease associated amyloids are often poorly soluble and structurally highly variable. This is known as structural polymorphism. To investigate to what extent molecular biophysics techniques can be standardized for studies of amyloid fibrils, five sites within the Horizon 2020 funded project MOlecular-Scale Biophysics Research Infrastructure (MOSBRI) addressed this issue via a joint research activity. For this study we selected bovine insulin as a convenient, accessible, and distributable amyloid model system. The benchmark comparable techniques for observing formed amyloid fibrils at the different sites were fluorescence spectroscopy of two amyloid ligands (ThT and pFTAA) and negative stain transmission electron microscopy. The outcome of this study yielded results with a large variability between different insulin amyloid fibril preparations, between sites, and within chemically identical preparations from one site. We mainly attribute these hard to control differences to the intrinsic polymorphic behaviour of insulin amyloid fibrils. We also present a range of experimental measurement techniques to highlight their potential use for studying amyloid structure and amyloid polymorphism.