Elongation kinetics of polyglutamine peptide fibrils: a quartz crystal microbalance with dissipation study

Abstract

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation.

Fig. 1

Proposed schema of polyQ fibril elongation. Shaded regions represent the original fibril. (a) Monomer must fully consolidate (“lock”) to the fibrillar structure before an additional monomer can add (“dock”). (b) Full consolidation of newly added monomer is not required to support further elongation. This results in changes to the structure of the fibril as it elongates. Ultimately, the β-sheet-rich structure of the monomer propagates through the fibril, but this is not prerequisite for further elongation. As sketched, the first three steps are addition only and the latter steps show slow consolidation. Under this mechanism, it would be possible to have simultaneous addition and consolidation, but with consolidation slower than addition.

Fig. 2

Transmission electron microscopy image of PG aggregates. Aggregates were prepared via freeze–thaw cycles of PG peptide in water adjusted to pH 3 with TFA. Image is representative of a large number of fields examined. The scale bar represents 200 nm.

Fig. 3

Immobilization of PG aggregates on SiO₂ surface and blocking of unoccupied sites with PLL. Vertical lines indicate changes in solvent conditions. Initially, water at pH 3 was pumped across the surface, followed by PG aggregates (in pH 3 water). The solution was then switched to Hepes buffer (pH 7); the slight shift in signal is likely due to the solvent switch. Finally, the sensor was exposed to PLL to block any remaining surface sites. (a) Mass associated. (b) Mass associated–dissipation plot for the period of peptide exposure.

Fig. 4

Effect of PG monomer concentration on elongation kinetics. Concentrations used were 10 μM (◯), 20 μM (■), and 40 μM (▲) in PBSA. Vertical lines indicate changes in solvent conditions. Results are representative of two independent experiments. (a) Mass associated. (b) Dissipation measurements. (c) Mass associated–dissipation plot for the period of peptide exposure.

Fig. 5

Effect of pH on PG elongation kinetics. Solutions of PG were prepared in pH 12 phosphate buffer at 20 μM (■) and 5 μM (◯). Vertical lines indicate times at which solutions were changed. Results are representative of two independent experiments. (a) Mass associated. Drift in the baseline is caused by ionization of the SiO₂ surface at pH 12. (b) Dissipation measurements. (c) Mass associated–dissipation plot for the period of peptide exposure.

Fig. 6

Effect of polyQ length on elongation kinetics. Solutions (20 μM) of monomeric Q8, Q20 (◯), or Q24 (■) in PBSA was exposed to a PG fibril-coated SiO₂ sensor. Vertical lines indicate times at which solutions were changed. Results are representative of two independent experiments. (a) Mass associated. (b) Dissipation measurements. (c) Mass associated–dissipation plot for the period of peptide exposure (Q8 not shown for clarity).

Fig. 7

Elongation kinetics of polyQ peptides at pH 12. Solutions (20 μM) of monomeric Q8, Q20 (◯), or Q24 (■) in a phosphate buffer adjusted to pH 12 was exposed to a PG fibril-coated SiO₂ sensor. Vertical lines indicate times at which solutions were changed. Results are representative of two independent experiments. (a) Mass associated. Drift in the baseline is caused by ionization of the SiO₂ surface at pH 12. (b) Dissipation measurements. (c) Mass associated–dissipation plot for the period of peptide exposure (Q8 not shown for clarity).

Fig. 8

Elongation kinetics of PP. Solutions (20 μM) of monomeric PP in PBS (◯) or pH 12 buffer (■) was exposed to a fibril-coated SiO₂ sensor. Results are representative of two independent experiments. Vertical lines indicate times at which solutions were changed. (a) Mass associated. Drift in the baseline is caused by ionization of the SiO₂ surface at pH 12. (b) Dissipation measurements. (c) Mass associated–dissipation plot for the period of peptide exposure.

Fig. 9

Extension of immobilized PG fibrils by aggregates in solution. Vertical lines indicate times at which solutions were changed. Unfiltered PG aggregates were diluted into PBS to a concentration of 20 μM and exposed to a PG fibril-coated SiO₂ sensor. Results are representative of two independent experiments. (a) Mass associated and dissipation measurements (secondary y-axis). (b) Mass associated–dissipation plot.

Fig. 10

Comparison of elongation kinetics as measured by OWLS and QCM-D. Solutions (20 μM) of monomeric Q20 or PG in PBS were exposed to a PG fibril-coated SiO₂ sensor. Vertical lines indicate times at which solutions were changed. (a) QCM-D data reproduced from Figs. 4 and 6. (b) OWLS data from PG and Q20. (c) Calculated density of the adlayer for PG (■) and Q20 (◯). Data from before the exposure of polyQ peptide have been removed for clarity. Results are representative of two independent experiments.