Effect of RNA on the supramolecular architecture of α-synuclein fibrils

Abstract

Structural changes associated with protein aggregation are challenging to study, requiring the combination of experimental techniques providing insights at the molecular level across diverse scales, ranging from nanometers to microns. Understanding these changes is even more complex when aggregation occurs in the presence of molecular cofactors such as nucleic acids and when the resulting aggregates are highly polymorphic. Infrared (IR) spectroscopy is a powerful tool for studying protein aggregates since it combines the label-free sensitivity to the cross-β architecture, an inherent feature of protein supramolecular aggregates, with the possibility to reach nanoscale sensitivity by leveraging atomic force microscopy (AFM)-assisted detection. Here, we present a combined approach that detects IR spectral markers of aggregation using various IR spectroscopy techniques, covering micro-to-nanoscale ranges, to study the effect of RNA on the supramolecular architecture of α-synuclein amyloid aggregates. We show a clear impact of RNA consistent with enhanced intermolecular forces, likely via a stronger hydrogen-bonded network stabilizing the cross-β architecture. AFM-assisted IR spectroscopy was crucial to assess that the more ordered the aggregates are, the stronger the structural impact of RNA. In addition, an RNA-induced reduction of the degree of polymorphism within the aggregate population is obtained.

Figure 1

Effect of RNA on the fibril formation kinetics and morphology of $\mathrm{\alpha }$S fibrils. (A) Left: AFM image of $\mathrm{\alpha }$S fibrils with a schematic representation of the two protofibrils that constitute the fibril (70). The $\mathrm{\beta }$-strands adopt a parallel and in-register arrangement and are perpendicular to the fibril axis (49). Right: schematics of the interstrand H-bond between the carbonyl and the amine groups in the peptide backbone, called cross-$\mathrm{\beta }$ architecture, detected by IR. (B) Amide-I band deconvolution of ${\mathrm{\alpha S}}_{\mathrm{WT}}$ fibrils (top) and oligomers (bottom) using five Lorentzian components centered at 1625, 1640, 1652, 1670, and 1690 ${\text{cm}}^{-1}$. Each Lorentzian component corresponds to a specific protein secondary structure based on its frequency position (41). (C) Experimental data from averaged time-resolved $\text{Proteostat}$™ fluorescence replicates of ${\mathrm{\alpha S}}_{103}$ in the absence (top) and presence (bottom) of RNA with standard deviations. (D) Maximum percentage values of the FTIR ($\mathrm{\beta }$-sheet)_P,AP component for the ${\mathrm{\alpha S}}_{103}$ (top) and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ (bottom) samples. See Figs. S2 and S3 for representative FTIR spectra and trend of the amide-I band deconvolution components. The data in (B) and (D) were fitted into a sigmoidal curve using the Hill function. Best-fit aggregation rates for fluorescence (and FTIR) are 1.3 $\pm$ 0.1 ${\mathrm{h}}^{-1}$ (1.5 $\pm$ 0.4 ${\mathrm{h}}^{-1}$) for ${\mathrm{\alpha S}}_{103}$ and 2.0 $\pm$ 0.2 ${\mathrm{h}}^{-1}$ (2.5 $\pm$ 1.3 ${\mathrm{h}}^{-1}$) for ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ samples. (E and F) CD and micro-FTIR absorption spectra (200 × 200 ${\mathrm{\mu m}}^2$ sample area) of ${\mathrm{\alpha S}}_{\mathrm{WT}}$ (continuous line) and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA}}$ (dashed line) at different sampling times. The micro-FTIR spectra are normalized at 1660 ${\text{cm}}^{-1}$. (G) Contact-mode AFM topography maps of ${\mathrm{\alpha S}}_{\mathrm{WT}}$ (top) and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA}}$ (bottom) fibrils. Scale bar, 200 nm. (H and I) Histograms of the height distribution of ${\mathrm{\alpha S}}_{\mathrm{WT}}$ (H) and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA}}$ (I) fibrils (bin size is 1 nm). In the inset, the trajectory of fibrils with different lengths (superimposed black lines in the AFM images of (G)), where the tangents at the initial end are aligned.

Figure 2

Effect of RNA on the supramolecular architecture of the less polymorphic ${\mathrm{\alpha S}}_{\mathrm{WT},\mathrm{seed}}$ fibrils. (A and B) Micro-FTIR absorption spectra (15 × 15 ${\mathrm{\mu m}}^2$) with the assignment of the secondary structure components (A) and corresponding second-order derivative curves (B) for the ${\mathrm{\alpha S}}_{\mathrm{WT},\mathrm{seed}}$ (purple line) and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA},\mathrm{seed}}$ (orange line). The inset of (B) shows representative second-order derivatives with Gaussian fits of the ($\mathrm{\beta }$-sheet)_P,AP component. The Gaussian fits have a central frequency at 1629.3 and 1626.7 ${\text{cm}}^{-1}$ and a FWHM of 5.4 and 4.3 ${\text{cm}}^{-1}$ for the ${\mathrm{\alpha S}}_{\mathrm{WT},\mathrm{seed}}$ and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA},\mathrm{seed}}$ curves, respectively. (C) Histogram of the ${\mathrm{\omega }}_{\mathrm{\beta }}$ distribution of ${\mathrm{\alpha S}}_{\mathrm{WT},\mathrm{seed}}$ (top) and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA},\mathrm{seed}}$ (bottom) fibrils measured in 15 × 15 ${\mathrm{\mu m}}^2$ areas (bin size is 0.1 ${\text{cm}}^{-1}$). Number of analyzed spectra is 73 (top) and 48 (bottom). In the insets, we show the average second-order derivatives from the low-frequency ($<$1626.5 ${\text{cm}}^{-1}$, purple and red line) and high-frequency ($>$1626.5 ${\text{cm}}^{-1}$, light purple and orange line) populations of the histograms. Arrows indicate the 1620 ${\text{cm}}^{-1}$ contribution. (D and E) PCA scores plots showing a clear separation between the ${\mathrm{\omega }}_{\mathrm{\beta }}$ of ${\mathrm{\alpha S}}_{\mathrm{WT},\mathrm{seed}}$ and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA},\mathrm{seed}}$ fibrils measured in 15 × 15 ${\mathrm{\mu m}}^2$ areas with an MCT detector (D) and 2.7 × 2.7 ${\mathrm{\mu m}}^2$ areas with an FPA detector (E). In the insets, the loadings plots show the PC1 and PC2 coordinates, which indicate the first and second principal components. PC1 and PC2 variance is 56% and 32% for FPA and 60% and 28% for MCT, respectively. (F) Sketch of the relative angle between the electric field in the tip-gold nanogap and the C=O cross-$\mathrm{\beta }$ dipole moment for a simplified case of an individual rod-like amyloid fibril (top) and an agglomerate of fibrils (bottom). (G) s-SNOM absorbance spectra (top) and corresponding second-order derivative curves (bottom) of ${\mathrm{\alpha S}}_{\mathrm{WT},\mathrm{seed}}$ (purple line) and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA},\mathrm{seed}}$ (orange line) fibril agglomerates. Spectra are the average of measurements at four different locations of the fibril agglomerates. (H) AFM topography maps of the ${\mathrm{\alpha S}}_{\mathrm{WT},\mathrm{seed}}$ (top) and ${\mathrm{\alpha S}}_{\mathrm{WT}+\mathrm{RNA},\mathrm{seed}}$ (bottom) fibril agglomerates where the s-SNOM spectra were acquired.

Figure 3

Effect of RNA on the morphology and supramolecular architecture of the highly polymorphic ${\mathrm{\alpha S}}_{103}$ aggregates. (A) Histogram of the ${\mathrm{\omega }}_{\mathrm{\beta }}$ distribution of ${\mathrm{\alpha S}}_{103}$ (top) and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ (bottom) fibrils measured in 15 × 15 ${\mathrm{\mu m}}^2$ areas (bin size is 0.1 ${\text{cm}}^{-1}$). Number of analyzed spectra is 79 (top) and 82 (bottom). (B) PCA scores plot showing very little separation between the ${\mathrm{\omega }}_{\mathrm{\beta }}$ of ${\mathrm{\alpha S}}_{103}$ and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ fibrils. In the inset, the loading plot shows the PC1 and PC2 coordinates, which indicate the first and second principal components. PC1 and PC2 variance is 51 and 42%, respectively. (C and D) AFM topography maps of ${\mathrm{\alpha S}}_{103}$ (C) and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ (D) agglomerate of amorphous aggregates and fibrils. (E) Second-order derivatives of AFM-IR spectra acquired on ${\mathrm{\alpha S}}_{103}$ (left) and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ (right) amorphous aggregates and fibrils. The dashed lines are the Gaussian fits of the cross-$\mathrm{\beta }$ negative peak. (F) Plot of the ${\mathrm{\omega }}_{\mathrm{\beta }}$ of the Gaussian fit versus the Gaussian FWHM for the ${\mathrm{\alpha S}}_{103}$ and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ amorphous aggregates and fibrils in (E). Circle markers show the average values corresponding to the ${\mathrm{\alpha S}}_{103}$ and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ samples. Horizontal and vertical error bars represent the standard deviation of the FWHM and ${\mathrm{\omega }}_{\mathrm{cross}-\mathrm{\beta }}$, respectively. The average ${\mathrm{\omega }}_{\mathrm{cross}-\mathrm{\beta }}$ values for ${\mathrm{\alpha S}}_{103}$ and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ are 1633 $\pm$ 1 ${\text{cm}}^{-1}$ and 1631 $\pm$ 1 ${\text{cm}}^{-1}$, respectively. Meanwhile, the average FWHM values are 7 $\pm$ 2 ${\text{cm}}^{-1}$ and 5 $\pm$ 2 ${\text{cm}}^{-1}$, respectively. The total number of analyzed AFM-IR spectra is 11 for the ${\mathrm{\alpha S}}_{103}$ and 12 for the ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ samples.

Figure 4

RNA has different structural effects on ${\mathrm{\alpha S}}_{103}$ aggregates with distinct conformations. (A and C) Second-order derivatives of AFM-IR spectra acquired on ${\mathrm{\alpha S}}_{103}$ and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ amorphous aggregates (A) and ordered fibrils (C). (B and D) AFM topography maps of ${\mathrm{\alpha S}}_{103}$ and ${\mathrm{\alpha S}}_{103+\mathrm{RNA}}$ amorphous aggregates (B) and fibrils (D) . Filled circle markers on the AFM maps indicate the location where the AFM-IR spectra were acquired.

Figure 5

RNA favors the formation of stronger H-bonding among the $\mathrm{\beta }$-sheets in the $\mathrm{\alpha }$S fibril structure. (A) Energy landscape of protein folding and aggregation. (B and C) Distribution of the low-energy levels related to fibril formation (top) and general chemical structure of $\mathrm{\beta }$-strands (bottom), inside the $\mathrm{\alpha }$S (B) and ${\mathrm{\alpha S}}_{+\mathrm{RNA}}$ (C) fibril, interacting by H-bonds. In the presence of RNA, the H-bond network is stronger (represented by thick dashed lines).