Conformational and aggregation properties of a PEGylated alanine-rich polypeptide

Abstract

The conformational and aggregation behavior of PEG conjugates of an alanine-rich polypeptide (PEG-c17H6) were investigated and compared to that of the polypeptide equipped with a deca-histidine tag (17H6). These polypeptides serve as simple and stimuli-responsive models for the aggregation behavior of helix-rich proteins, as our previous studies have shown that the helical 17H6 self-associates at acidic pH and converts to β-sheet structures at elevated temperature under acidic conditions. In the work here, we show that PEG-c17H6 also adopts a helical structure at ambient/subambient temperatures, at both neutral and acidic pH. The thermal denaturation behavior of 17H6 and PEG-c17H6 is similar at neutral pH, where the alanine-rich domain has no self-association tendency. At acidic pH and elevated temperature, however, PEGylation slows β-sheet formation of c17H6, and reduces the apparent cooperativity of thermally induced unfolding. Transmission electron microscopy of PEG-c17H6 conjugates incubated at elevated temperatures showed fibrils with widths of ∼20-30 nm, wider than those observed for fibrils of 17H6. These results suggest that PEGylation reduces β-sheet aggregation in these polypeptides by interfering, only after unfolding of the native helical structure, with interprotein conformational changes needed to form β-sheet aggregates.

Figure 1

CD spectra of 50 μM PEG5K-c17H6 in pH 2.3 buffer during (A) unfolding upon increasing temperature from 5°C to 80°C and (B) refolding upon decreasing temperature from 80°C to 5°C subsequent to the incubation at 80°C for 3 h.

Figure 2

Comparison of CD melting curves of 17H6 (circles), PEG5K-c17H6 (squares), and PEG10K-c17H6 (triangles) at pH 2.3 (C = 50 μM).

Figure 3

Comparison of the first DSC scans of 17H6 (solid line), PEG5K-c17H6 (dashed line), and PEG10K-c17H6 (dotted line) in pH 2.3 buffer (C = 100 μM).

Figure 4

Consecutive DSC scans of PEG5K-c17H6 in pH 2.3 buffer. The first scan is given as dotted line; the other scans are represented as solid line.

Figure 5

CD spectra, collected at 1 h intervals upon incubation at 80°C for 18 h, of 50 μM solutions of (A) PEG5K-c17H6, (B) PEG10K-c17H6, in pH 2.3 buffer.

Figure 6

CD spectra obtained upon cooling of solutions of (A) PEG5K-c17H6, (B) PEG10K-c17H6 from 80°C to 5°C, at 5°C temperature intervals, subsequent to the incubation at 80°C for 18 h.

Figure 7

Comparison of the change in [θ]_MRE,205 over time, with incubation at 80°C, of 17H6 (circles), PEG5K-c17H6 (squares), and PEG10K-c17H6 (triangles).

Figure 8

TEM images of PEG5K-c17H6 at pH 2.3, after incubation at 80°C for 18 h. Scale bars: (A) 500 nm, (B) 100 nm.

Figure 9

TEM images of PEG10K-c17H6 at pH 2.3, after incubation at 80°C for 18 h. Scale bars: (A) 200 nm, (B) 100 nm.