Aggregation gatekeeper and controlled assembly of Trpzip β-hairpins

Abstract

Protein and peptide aggregation is an important issue both in vivo and in vitro. Herein, we examine the aggregation behaviors of two well-studied β-hairpins, Trpzip1 and Trpzip2. Previous studies suggested that Trpzip2 remains monomeric up to a concentration of ~15 mM whereas Trpzip1 readily aggregates at micromolar concentrations at acidic or neutral pH. This disparity is puzzling considering that these two peptides differ only in their turn sequences (i.e., GN vs NG). We hypothesize that these peptides can aggregate from their folded states via native edge-to-edge interactions and that the Lys8 residue in Trpzip2 is a more effective aggregation gatekeeper, because of a more favorable orientation. In support of this hypothesis, we find that increasing the pH to 13 or replacing Lys8 with a hydrophobic and photolabile Lys analogue, Lys(nvoc), leads to a significant increase in the aggregation propensity of Trpzip2, and that the aggregation of this Trpzip2 mutant can be reversed upon restoring the native Lys side chain via photocleavage of the nvoc moiety. In addition, we find that while both Trpzip1 and Trpzip2 form parallel β-sheet aggregates, the Lys(nvoc) Trpzip2 mutant forms antiparallel β-sheets and more stable fibrils. Taken together, these findings provide another example showing how sensitive peptide and protein aggregation is to minor sequence variation and that it is possible to use a photolabile non-natural amino acid, such as Lys(nvoc), to tune the rate of peptide aggregation and to control fibrillar structure.

Figure 1

NMR structures of Trpzip1 (PDB entry 1LE0) and Trpzip2 (PDB entry 1LE1), as indicated, and the proposed dimerization scheme, showing the difference in the orientations of the Lys8 side chains.

Figure 2

Amide I′ spectra of Trpzip1 at different concentrations, as indicated. Shown in the inset is the amide I′ band of Trpzip2 at 10 mM. These data were collected after the peptide samples had been incubated for 24 h at 25 °C.

Figure 3

Intensity of the 1616 cm^–1 band of Trpzip1 (1.2 mM, pH 3) as a function of incubation time, showing the aggregation kinetics of this peptide at acidic pH. For comparison, the aggregation data of Trpzip1 (0.7 mM) obtained at pH 13 are shown in the inset. The corresponding FTIR spectra are presented in Figure S2 of the Supporting Information.

Figure 4

Amide I′ spectra of Trpzip2 (∼6 mM) at pH 13 measured after the peptide sample had been incubated for 1 and 6 days, as indicated.

Figure 5

Representative amide I′ spectra of Trpzip2-K (0.5 mM, pH 3) obtained after different sample incubation times, as indicated. Shown in the inset is the intensity of the 1616 cm^–1 band as a function of incubation time. The band intensities were obtained from the FTIR spectra shown in Figure S5 of the Supporting Information.

Figure 6

CD spectra of monomeric and aggregated Trpzip2-K samples (40 μM, pH 3), as indicated. The aggregated sample was prepared by diluting a more concentrated peptide sample (0.5 mM) that had been incubated for 14 days to allow aggregate formation.

Figure 7

Amide I′ bands of an aggregated Trpzip2-KK sample (2.2 mM, D₂O) obtained under different conditions, as indicated. These spectra show that photocleavage of the nvoc group on Lys8 results in aggregate disassembly. The band located near 1700 cm^–1 arises from the C=O stretching vibration of the Lys12 side chain acrylamide.

Scheme 1. Acylation of the Primary Amine of Lys12 in Trpzip2-K

The resulting peptide is termed Trpzip2-KK.

Figure 8

Amide I′ bands of Trpzip2-W4A (10 mM, pH 3) obtained at two incubation times, as indicated.

Figure 9

Amide I′ band of Trpzip1 (2.4 mM, pH 3) as a function of temperature.

Figure 10

Amide I′ band of Trpzip2-K (4 mM, pH 3) as a function of temperature.

Figure 11

Representative AFM image of the peptide fibrils formed by Trpzip2-K after an incubation period of 14 days.

Figure 12

Representative AFM image of the peptide fibrils and aggregates formed by Trpzip1 after an incubation period of 14 days.