Insulin fibril nucleation: the role of prefibrillar aggregates

Abstract

Dynamic light scattering and Fourier transform infrared spectroscopy were used to study the formation of prefibrillar aggregates and fibrils of bovine pancreatic insulin at 60 degrees C and at pH 1. The kinetics of disintegration of the prefibrillar aggregates were also studied using these techniques after a quench to 25 degrees C. These experiments reveal that formation of prefibrillar aggregates is reversible under the solution conditions studied and show that it is possible to significantly reduce the nucleation (lag) times associated with the onset of fibril growth in bovine pancreatic insulin solutions by increasing the concentration of prefibrillar aggregates in solution. These results provide convincing evidence that less structured prefibrillar aggregates can act as fibril-forming intermediates.

FIGURE 1

Particle size distributions obtained from DLS studies of prefibrillar aggregates in solutions of BPI (20 mg mL⁻¹, pH 1) after a temperature quench from 60°C to 25°C. Data were collected at times of 150 min (dotted line), 500 min (dashed line), and 2940 min (solid line) after the quench. All size distributions are normalized in such a way that the size of the peak centered at ∼13 nm is equal to unity.

FIGURE 2

Light scattering and FTIR studies of the disintegration of prefibrillar aggregates in solutions of BPI (20 mg mL⁻¹, pH 1) after a temperature quench from 60°C to 25°C. Data are shown for the decay in the total light scattering intensity (▪, DLS) and the area of the ratioed β-sheet peak (○, FTIR, see text) as a function of time. The inset shows an AFM image of clusters of prefibrillar aggregates taken over a 5 μm × 5 μm area. The scale bar on this image corresponds to a distance of 1 μm.

FIGURE 3

FTIR spectra collected during the disintegration of prefibrillar aggregates in solutions of BPI (20 mg mL⁻¹, pH 1) after a temperature quench from 60°C to 25°C. Data are shown for spectra collected at times of 5 (○), 60 (□), 150 (▵), 500 (•), and 2100 min (▪) after the quench. The solid line is a spectrum taken from freshly dissolved insulin at 25°C.

FIGURE 4

Fibril nucleation studies in BPI solutions (20 mg mL⁻¹, pH 1) with different thermal histories. Data are shown for the ratioed β-sheet growth kinetics obtained from FTIR spectroscopy measurements of BPI solutions (see text). The data were collected from freshly dissolved BPI solutions that were incubated at 60°C (•), as well as solutions that were heated to 60°C for 60 min before being quenched to 25°C and then reheated to 60°C within 5 min (▵) and held at 25°C for 120 h before being reheated to 60°C (○). The solid lines shown in this figure mark the position of the plateau corresponding to the end of prefibrillar aggregate formation and the slopes of the regions corresponding to the period of fibril growth (13), respectively. The nucleation (lag) times for fibril growth can be determined from the times at which the fibril growth slopes intersect the plateau line. The horizontal error bars represent the uncertainty in the fibril nucleation times obtained in each set of experiments and are shown on a single data point in each set for reasons of clarity.