Fig. 2: Nanomolar affinity IAPP/ACM interactions yield amyloid fibril-resembling but ThT-invisible nanofibers.

a Nanomolar affinity IAPP/ACM interactions as determined by fluorescence spectroscopy. Fluorescence spectra of Fluos-IAPP (5 nM) and its mixtures with Nle3-VF (pH 7.4) at indicated molar ratios (data from one representative binding assay from n = 3 independent assays). Inset, binding curve; data are means ± SD from n = 3 independent assays. b IAPP/ACM interactions result in hetero-dimers and medium-to-high MW hetero-assemblies. Characterization of IAPP/Nle3-VF hetero-complexes via cross-linking at different time points and NuPAGE and Western blot using anti-IAPP (left) or anti-Aβ (right) antibodies. IAPP/Nle3-VF mixtures (1/2; IAPP, 30 µM) were cross-linked with glutaraldehyde; the orange box indicates medium-to-high MW hetero-assemblies (major species); arrows indicate hetero-di-/-tri-/tetramers. Representative results from >3 independent assays. c Characterization of IAPP/Nle3-VF hetero-assemblies via size exclusion chromatography (SEC). Chromatograms of IAPP alone (16.5 µM) or its mixtures with Nle3-VF (1/2) at 0 h and at 96 h (mAU, milli-absorbance units). The black arrow indicates IAPP monomers and Nle3-VF dimers; blue arrows indicate IAPP/Nle3-VF hetero-dimers and medium-to-high MW hetero-assemblies. Similar results were found in two independent assays. d IAPP/Nle3-VF co-assemblies are more disordered than β-sheet-rich IAPP assemblies. Far-UV CD spectra of 7-day-aged IAPP (16.5 µM; pH 7.4) and its mixtures (1/2) with the Nle3-VF or VGS-VF (non-inhibitor) are shown; for comparison, the spectrum of freshly dissolved IAPP (0 h) is also shown. CD spectra are the average of three spectra of the same solution. e IAPP/Nle3-VF co-assembly blocks surface-exposure of hydrophobic clusters occurring at early steps of IAPP amyloid self-assembly as determined by anilinonaphthalene 8-sulfonate (ANS) binding. Fluorescence emission spectra of ANS alone/with IAPP (2 µM) (left) and of ANS alone/with IAPP/ACM (1/2) mixtures (right) (pH 7.4) were measured at various time points of self- or co-assembly as indicated (representative results from two independent assays). f IAPP/ACM interactions result in ThT-invisible fibrils of indistinguishable appearance to fIAPP by TEM. TEM images of 7-day-aged IAPP (16.5 µM) and its mixtures (1/2) with ACMs or VGS-VF (non-inhibitor) (solutions from Fig. 1d, f). Scale bars: 100 nm. Representative images from seven independent assays for fIAPP and the IAPP/Nle3-VF mixture and two to three similar independent assays for the other IAPP/ACM mixtures. g fIAPP and fibrils in IAPP/Nle3-VF mixture exhibit the amyloid cross-β structure signature. X-ray fiber diffraction patterns of fIAPP and fibrils present in aged IAPP/Nle3-VF mixture (1/2) showed major meridional and equatorial reflections at ~4.7 and ~10 Å. Data were representative of two independent experiments.