UV resonance Raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization

Abstract

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D(2)Q(10)K(2) sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a β-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) Methods Enzymol.413, 34-74] to dissolve the Q10 β-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5(1)-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 β-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5(1)-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.

Figure 1

Temperature dependence of the 204 nm excited UVRR spectra of valeramide in water at 22 °C (Black) and 65 °C (Red). Water contributions were removed. The intensities were normalized to the 932 cm⁻¹ ClO₄⁻ peak height.

Figure 2

The 204 nm excited UVRR spectra of valeramide at 22 °C: Black in water; Red in pure acetonitrile. Solvent contributions were removed. The intensities were normalized to the AmIII+δ_CH2 peak height.

Figure 3

The 204 nm excited UVRR spectra of valeramide solid (Red) and in water (Black) at 22 °C. Water contributions were removed. The intensities were normalized to the AmIII+δ_CH2 peak height.

Figure 4

CD spectra of 1 mg/ml DQ10 (solid line) and NDQ10 (dashed line) in pure water at 22 °C. Measured by using a 0.02 cm path length cuvette.

Figure 5

The 204 nm excited UVRR spectra of the DQ10 (black) and NDQ10 (red) in pure water at 22 °C. ^b indicates backbone vibration; ^s indicates side chain vibration. The intensities were normalized to the AmIII^b peak height.

Figure 6

Calculated Ψ-angle distributions for the NDQ10 and DQ10 in pure water at 22 °C.

Figure 7

The 198 (blue) and 204 nm (black) excited UVRR spectra of the NDQ10 in pure water at 22 °C, and the difference spectrum between them (red). The intensities were normalized to the AmIII^b peak height before spectral subtraction.

Figure 8

The difference spectra between the 198 and 204 nm excited UVRR spectra of the NDQ10 (red) and DQ10 (black) in pure water at 22 °C. The 204 nm excited UVRR spectrum of glutamine in pure water at pH 1.6 at 22 °C (blue).

Figure 9

The 204 nm excited UVRR spectra of the NDQ10 solid (black) and solution (red), DQ10 solid (blue) and solution (green) at 22 °C. ^b indicates backbone vibration; ^s indicates side chain vibration. The intensities were normalized to the AmIII^b peak height.

Figure 10

Powder x ray diffraction of NDQ10 solid. The sample was prepared by slowly evaporating NDQ10 solution on a glass slide over ~ 2 days.

Figure 11

Electron micrographs of (a) NDQ10 fibrils in pure water after incubation at 60 °C for ~ 1 week and (b) DQ10 fibrils in pure water upon seeding with 2% NDQ10 solution after incubation at 60 °C for ~ 4 days. (c) 204 nm excited UVRR spectra of the NDQ10 fibrils, the DQ10 fibrils and the NDQ10 solution at 22 °C.

Figure 12

Proposed structures of NDQ10 and DQ10. NDQ10 occurs as β-sheets in which the GLN side chains form HB to the backbone or other GLN side chains. DQ10 adopts PPII and 2.5₁-helix conformations in which the GLN side chains form HB to water. Main chain – main chain HB a, main chain – side chain HB b, side chain – side chain HB c.