Examining polyglutamine peptide length: a connection between collapsed conformations and increased aggregation

Abstract

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, of which the best known is Huntington's. Expansion of the polyglutamine domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. The age of onset of disease is inversely correlated with the length of the polyglutamine domain; this correlation motivates an examination of the role of the length of the domain on aggregation. In this investigation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides lacked secondary structure. Fluorescence resonance energy transfer studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. The effective persistence length was estimated to decrease from approximately 11 to approximately 7 A as the number of glutamines increased from 8 to 24. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20, and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate-buffered saline at 37 degrees C. Thus, Q16 stands at the transition point between good and poor solvent and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature.

Figure 1. CD spectra of polyQ peptides

Peptide stock solutions were diluted into a pH 7.4 phosphate buffer, (10 mM buffer salts, 140 mM NaF) to a concentration of 20 μM peptide, and filtered through a 0.45 μm membrane directly into a cuvette. Data was normalized to per residue molar ellipticity.

Figure 2. Length of polyQ region determined by FRET

Fluorescence spectra of peptides with and without the fluorescence acceptor dansyl were collected and used to calculate the distance between the fluorophores R. Peptides were diluted to 5 μM in pH 7.4 PBSA (□) or pH 12 buffer (●). Readings were taken in duplicate, and error bars indicate the standard deviation of the measurement. (a): Average length of polyQ region determined by FRET. (b) Persistence length, l_p.

Figure 3. Dynamic light scattering hydrodynamic radius of polyQ peptides

Disaggregated peptide stock solution was diluted to a concentration of 20μM in PBSA, then immediately filtered directly into a light scattering cuvette and held at 37°C. Apparent z-averaged hydrodynamic radius R_hz, as determined from cumulants analysis of autocorrelation data collected at 90° scattering angle. Data shown for Q16 (■), Q20 (○), and Q24 (▲). Q8 and Q12 did not scatter above background.

Figure 4. Scattered light intensity of polyQ peptides

Data shown for Q16 (■), Q20 (○), and Q24 (▲). Q8 and Q12 did not scatter above background. (a) Intensity of scattered light at 90°. Scattering due to the solvent was subtracted and results were normalized to the scattering intensity of toluene to account for changes in laser strength and aperture and to the mass concentration of peptide. (b) Total intensity of scattered light over a longer period of time.

Figure 5. TEM image of polyQ peptide aggregates

Samples were prepared at 20 μM in PBSA, and then incubated at 37°C prior to imaging. Images are representative of a large number of fields examined. The length of the white bar is 200 nm. (a) Q20, 3 hours incubation. (b) Q24, 3 hours incubation. (c) Q20, 40 days incubation. (d) Q16, 40 days incubation.

Figure 6. Sedimentation kinetics of polyQ peptides

Peptides [Q8 (△), Q12 (□), Q16 (■), Q20 (○), and Q24 (▲)] were diluted into PBSA at 20 μM, aliquoted into tubes, and incubated at 37°C. At the times indicated, a tube was centrifuged and the peptide concentration in the supernatant was determined using a BCA assay. Data is reported as the wt% peptide soluble, calculated from the ratio of the supernatant to the total concentration in an uncentrifuged sample. Lines are drawn as an aid to the eye.

Figure 7. Hydrodynamic radius of polyQ peptides from wormlike chain model

R_h was calculated from the Yamakawa-Fujii equations for a wormlike chain, assuming a chain diameter of 4 Å and a contour chain length (Å) of 3.8N = 3.8(N_Q + 6), where N = N_Q + 6 accounts for the number of glutamines in the peptide plus 6 flanking residues. Results are shown for l_p = 5 Å (□, ν= 0.37), l_p = 12 Å (○, ν= 0.44), and l_p = 22 N_Q ^−0.36 (●, ν= 0.30).

Figure 8. Schematic illustrating proposed kinetic pathway

Phase 1 consists of disordered peptides, with longer peptides showing more collapse. In phase 2, peptides with Q ≥ 16 associate in a nonspecific manner, leading to the amorphous aggregates present in phase 3. The amount of aggregated material in phase 3 is proportional to the number of glutamines. In phase 4, Q24 forms fully mature, structured, insoluble aggregates, while Q16 failed to sediment in 35 days.