Modulation of self-association and subsequent fibril formation in an alanine-rich helical polypeptide

Abstract

Thermal unfolding, reversible self-association, and irreversible aggregation were investigated for an alanine-rich helical polypeptide, 17-H-6, with sequence [AAAQEAAAAQAAAQAEAAQAAQ] 6. Dynamic light scattering, transmission electron microscopy, and thermal unfolding measurements indicate that 17-H-6 spontaneously and reversibly self-associates at acidic pH and low temperature. The resulting multimers have a compact, globular morphology with an average hydrodynamic radius approximately 10-20 nm and reversibly dissociate to monomers upon an increase to pH 7.4. Both free monomer and 17-H-6 chains within the multimers are alpha-helical and folded at low temperature. Reversible unfolding of the monomer occurs upon heating of solutions at pH 7.4. At pH 2.3, heating first causes incomplete dissociation and unfolding of the constituent chains. Further incubation at elevated temperature induces additional structural and morphological changes and results in fibrils with a beta-sheet 2 degrees structure and a characteristic diameter of 5-10 nm (7 nm mean). The ability to modulate association and aggregation suggests opportunities for this class of polypeptides in nanotechnology and biomedical applications.

Figure 1

Full wavelength spectra of 17-H-6 during (a) unfolding and (b) refolding at 50 μM polypeptide in pH 7.4 PBS. In each panel, spectra are shown for 5-80 °C in 5 °C increments.

Figure 2

Full wavelength spectra of 17-H-6 during (a) unfolding and (b) refolding at 50 μM polypeptide in pH 2.3 phosphate with 150 mM salt. In each panel, spectra are shown for 5-80 °C in 5 °C increments.

Figure 3

Time course of random coil-β-sheet transition of 50 μM 17-H-6 at pH 2.3 at 80 °C.

Figure 4

CD melting curves for 17-H-6 in pH 7.4 PBS buffer.

Figure 5

Characterization of the thermodynamic parameters of unfolding for 17-H-6 at pH 2.3. (a) CD melting curves and (b) two-state model thermodynamic parameters of 17-H-6 at pH 2.3; T_m (closed circles) and ΔH_VH (open circles).

Figure 6

Comparison of DSC curves of 100 μM 17-H-6 at different pH values; pH 7.4 (solid line) and pH 2.3 (dashed line).

Figure 7

Consecutive DSC scans of 100 μM 17-H-6 in pH 2.3 buffer.

Figure 8

Representative plots of (a) change in autocorrelation function as a function of delay time represented by experimental data points (circles) and cumulant fit (solid line) and (b) angular dependence of relaxation time represented by experimental data points (circles) and linear fit (solid line) (C = 50 μM).

Figure 9

TEM pictures of room temperature aggregates of 17-H-6 at pH 2.3. Scale bars: 100 nm.

Figure 10

TEM pictures of 100 μM of 17-H-6 at pH 2.3 annealed at 80 °C for 18 h. Scale bars: (a) 200 nm (b) 50 nm.

Figure 11

Schematic depiction of unfolding, self-association, and aggregation for 17-H-6. The upper portion of the scheme shows simple monomer unfolding and is operative at pH conditions where the Glu side chains are charged and monomer-monomer interactions are therefore repulsive. The remainder of the scheme applies at low pH, where Glu side chains are uncharged, and monomer-monomer interactions are significantly attractive even for folded molecules.