Unraveling VEALYL Amyloid Formation Using Advanced Vibrational Spectroscopy and Microscopy

Abstract

Intermediate species are hypothesized to play an important role in the toxicity of amyloid formation, a process associated with many diseases. This process can be monitored with conventional and two-dimensional infrared spectroscopy, vibrational circular dichroism, and optical and electron microscopy. Here, we present how combining these techniques provides insight into the aggregation of the hexapeptide VEALYL (Val-Glu-Ala-Leu-Tyr-Leu), the B-chain residue 12-17 segment of insulin that forms amyloid fibrils (intermolecularly hydrogen-bonded β-sheets) when the pH is lowered below 4. Under such circumstances, the aggregation commences after approximately an hour and continues to develop over a period of weeks. Singular value decompositions of one-dimensional and two-dimensional infrared spectroscopy spectra indicate that intermediate species are formed during the aggregation process. Multivariate curve resolution analyses of the one and two-dimensional infrared spectroscopy data show that the intermediates are more fibrillar and deprotonated than the monomers, whereas they are less ordered than the final fibrillar structure that is slowly formed from the intermediates. A comparison between the vibrational circular dichroism spectra and the scanning transmission electron microscopy and optical microscope images shows that the formation of mature fibrils of VEALYL correlates with the appearance of spherulites that are on the order of several micrometers, which give rise to a "giant" vibrational circular dichroism effect.

Figure 1

1D-IR (FTIR) spectra of 11 mM VEALYL solution after the pH is lowered from 7 to 2.5. The spectra are first background subtracted with a buffer-only spectrum and then normalized with respect to the amide-I area of the first spectrum. The decreasing, broad ∼1645 cm⁻¹ peak indicates decreasing amounts of random coil structures, which are transformed into intermolecular β-sheets, as indicated by the increasing peaks at ∼1622 and ∼1682 cm⁻¹. To see this figure in color, go online.

Figure 2

SVD analysis of the time-dependent FTIR spectra of aggregating VEALYL. For the first four components, the left- and right-singular vectors are depicted in blue, green, orange, and red, respectively. To aid the analysis, a spectrum measured at t = 5250 min (data not shown) was included in the data set to include a time point at which the aggregation process was fully completed. To see this figure in color, go online.

Figure 3

Feasible spectra (left column) and associated concentration profiles (right column). To see this figure in color, go online.

Figure 4

2D-IR spectra of VEALYL after the pH of the solution has been lowered from 7 to 2.5. The increasing signal strength at ∼1622 and 1682 cm⁻¹ indicates that amyloid β-sheets are formed, and the decreasing inverted slope of the modes with time indicates that the heterogeneity is decreasing. The spectrum labeled “44 h^∗” is measured at a spot in the IR sample cell ∼500 μm away from the four preceding spectra (all of a 11.75 mM VEALYL sample). The mature fibril is measured of a 26.5 mM solution that was incubated for 5 days. We did not observe a strong dependency of the spectral line shape once the concentration threshold for aggregation was exceeded. To see this figure in color, go online.

Figure 5

VCD spectra of 11 mM VEALYL after the pH of the solution has been lowered from 7 to 2.5, with tentative assignments indicated. The increasing signal strength at ∼1621 and 1635 cm⁻¹ indicates that chiral structures are formed that are of the order of 6 μm (the wavelength equivalent of ∼1650 cm⁻¹). To see this figure in color, go online.

Figure 6

The maximum amplitude of the VCD signal at 1622 cm⁻¹ versus time, fitted with the standard Avrami equation with n = 2.5. To see this figure in color, go online.

Figure 7

An overview of the data, analysis, and interpretation of the vibrational spectroscopy and imaging techniques used to study the amyloid formation of VEALYL. Row 1 shows the qualitative description and artist impressions of the structural interpretations of the various aggregation phases. Row 2 shows the 1D-IR (FTIR) spectra. Row 3 shows the 2D-IR spectra. Row 4 shows the VCD spectra. Row 5 shows the normalized profiles of the intensity at VEALYL’s amyloid β-sheet frequency (1622 cm⁻¹) for the various techniques and concentration profiles of the three species from the FACPACK analysis of the 1D-IR data. Row 6 shows the light microscope images. Row 7 shows the STEM images. The 2D-IR spectra were recorded only up to 44 h of the same sample due to experimental procedural reasons (see main text). The third and fourth STEM images are recorded of the same sample with a VCD intensity that is 50% of the maximum, and an amyloid β-sheet IR signal that is already at its maximum because the different species (as shown in the fifth row) coexist when the intermediates and the mature fibrils are formed. To see this figure in color, go online.