Nanoscopic and Photonic Ultrastructural Characterization of Two Distinct Insulin Amyloid States

Abstract

Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.

Factors affecting the physical stability (aggregation) of peptide therapeutics

“…In addition, small amounts of deamidated material can induce amyloid formation of the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for aggregation studies [58,[221][222][223][224][225][226][227].…”

Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™ stains

“…Some of these are referred to as luminescent conjugated oligothiophenes (LCOs) [ 68 – 76 ] and are marketed as Amytracker™ 480 and 680 (derived, respectively, from HS163 and HS169 in the literature [ 71 , 74 ]), but proprietary structures that are not identical to them; figure 1 for the chemical structures of HS163 and HS169). Although they too show strong selectivity for, and enhanced fluorescence when binding to, classical amyloid proteins, their staining properties clearly differ from those of ThT [ 68 , 70 , 79 – 81 ]. In some cases, their binding affects prion formation itself [ 82 , 83 ] (and even ThT has anti-ageing properties at low concentrations [ 84 ]).…”

Thioflavine-T and Congo Red Reveal the Polymorphism of Insulin Amyloid Fibrils When Probed by Polarization-Resolved Fluorescence Microscopy

“…Although it does not reach the power of structural analysis techniques, fluorescent microscopy is an established technique, especially for diagnostic purpose, because it allows to observe easily the formation of amyloid fibrils in tissues or in vitro 7,8,9,10,11 . In this context, much work has been carried out in order to synthesize efficient fluorescent probes that bind specifically to the amyloid structure 12,13,14 .…”