Aggregation of proteins with expanded glutamine and alanine repeats of the glutamine-rich and asparagine-rich domains of Sup35 and of the amyloid beta-peptide of amyloid plaques

Abstract

The exon-1 peptide of huntingtin has 51 Gln repeats and produces the symptoms of Huntington's disease in transgenic mice. Aggregation of the yeast Sup35 protein into prions has been attributed to its glutamine-rich and asparagine-rich domain. Here, we show that poly-L-asparagine forms polar zippers similar to those of poly-L-glutamine. In solution at acid pH, the glutamine-rich and asparagine-rich 18-residue Sup35 peptide, rendered soluble by the addition of two aspartates at the amino end and two lysines at the carboxyl end, gives a beta-sheet CD spectrum; it aggregates at neutral pH. A poly-alanine peptide D(2)A(10)K(2) gives an alpha-helical CD spectrum at all pHs and does not aggregate; a peptide with the sequence of the C-terminal helix of the alpha-chain of human hemoglobin, preceded by two aspartates and followed by two lysines, exhibits a random coil spectrum and does not aggregate either. Alignment of several beta-strands with the sequence of the 42-residue Alzheimer's amyloid beta-peptide shows that they can be linked together by a network of salt bridges. We also asked why single amino acid replacements can so destabilize the native structures of proteins that they unfold and form amyloids. The difference in free energy of a protein molecule between its native, fully ordered structure and an amorphous mixture of randomly coiled chains is only of the order of 10 kcal/mol. Theory shows that destabilization of the native structure by no more than 2 kcal/mol can increase the probability of nucleation of disordered aggregates from which amyloids could grow 130,000-fold.

Figure 1

X-ray fiber diffraction diagrams of the peptide D₂Q₁₅K₂ (Left) and of the exon-1 protein of huntingtin with 51 Gln repeats.

Figure 2

Far-UV CD spectra plotting the mean residue ellipticity against wavelength for (A) the poly-l-asparagine peptide D₂N₁₅K₂ and (B) the yeast prion protein Sup35 peptide. The spectra are typical of pure β-sheet at pH 2 but suggest a decrease of β-sheet with increasing pH to neutral pH. The signal for the yeast prion protein Sup35 peptide eventually becomes that of a random coil at the highest pH of 10.4.

Figure 3

Light-scattering experiments on the yeast prion protein Sup35 peptide indicate aggregation that peaks between pH 5.5 and 7.5. This effect is negligible below pH 3.0 and above pH 9.0. (The signal from the hemoglobin peptide rose to the equivalent of eight light-scattering units at pH 7–7.5, too small to show on the plot.)

Figure 4

The polyalanine peptide D₂A₁₀K₂ folds to give an α-helix type far-UV CD spectrum. The plot for α-helix at pH 3.5 is weaker than at pH 4.4 and increases slightly with pH to pH 8.3.

Figure 5

The C-terminal α-helix peptide of hemoglobin exhibits a random coil structure in the far-UV CD. There are no effects on folding with changes in pH.

Figure 6

Alignment of the sequence of the 42-residue peptide of the plaques (10) of Alzheimer's disease. The red boxes mark salt bridges between acids and bases and a hydrogen bond between a Tyr and an Asn; the blue circles mark uncompensated changes.