VCD spectroscopic properties of the beta-hairpin forming miniprotein CLN025 in various solvents

Abstract

Electronic and vibrational circular dichroism are often used to determine the secondary structure of proteins, because each secondary structure has a unique spectrum. Little is known about the vibrational circular dichroic spectroscopic features of the beta-hairpin. In this study, the VCD spectral features of a decapeptide, YYDPETGTWY (CLN025), which forms a stable beta-hairpin that is stabilized by intramolecular weakly polar interactions and hydrogen bonds were determined. Molecular dynamics simulations and ECD spectropolarimetry were used to confirm that CLN025 adopts a beta-hairpin in water, TFE, MeOH, and DMSO and to examine differences in the secondary structure, hydrogen bonds, and weakly polar interactions. CLN025 was synthesized by microwave-assisted solid phase peptide synthesis with N(alpha)-Fmoc protected amino acids. The VCD spectra displayed a (-,+,-) pattern with bands at 1640 to 1656 cm(-1), 1667 to 1687 cm(-1), and 1679 to 1686 cm(-1) formed by the overlap of a lower frequency negative couplet and a higher frequency positive couplet. A maximum IR absorbance was observed at 1647 to 1663 cm(-1) with component bands at 1630 cm(-1), 1646 cm(-1), 1658 cm(-1), and 1675 to 1680 cm(-1) that are indicative of the beta-sheet, random meander, either random meander or loop and turn, respectively. These results are similar to the results of others, who examined the VCD spectra of beta-hairpins formed by (D)Pro-Xxx turns and indicated that observed pattern is typical of beta-hairpins.

Figure 1

The backbone and side chains of the crystal structure of CLN025. Random meander is cyan, β-sheet is red, turn is green and the H-bonds are represented by yellow dotted lines.

Figure 2

The ECD spectra of 100 µM CLN025 in 20 mM potassium phosphate buffer at 0 °C (violet), 20 °C (blue), 40 °C (green), 60 °C (orange) and 80 °C (red) and cooled from 80 °C to 0 °C (black).

Figure 3

The ECD spectra of 100 µM CLN025 in 20 mM potassium phosphate buffer (black), TFE (red) and MeOH (blue) at 20 °C.

Figure 4

Normalized VCD (top) and IR absorption (bottom) spectra of 20 mg mL⁻¹ CLN025 in 5% (v/v) DMSO-d₆ in 20 mM deuterated potassium phosphate buffer (black), TFE-d (red), MeOH-d₄ (blue) and DMSO-d₆ (green). The deconvoluted absorption spectra are shown by the thin curves.

Figure 5

Normalized VCD (top) and IR absorption (bottom) spectra of 20 mg mL⁻¹ CLN025 in 5% (v/v) DMSO-d₆ in 20 mM deuterated potassium phosphate buffer at 5 °C (black), 20 °C (blue) and 60 °C (red).

Figure 6

The backbone and side chains of the middle structure of the most populated cluster of trajectories of MD simulations of CLN025 in A, H₂O 5 °C; B, H₂O 27 °C; C, H₂O 60 °C; D, TFE; E, MeOH and F, DMSO. Random meander is cyan, β-sheet is red, turn is green and the H-bonds are yellow dotted lines.

Figure 7

DSSP analysis of the trajectories of simulations of CLN025 in A, H₂O 5 °C; B, H₂O 27 °C; C, H₂O 60 °C; D, TFE; E, MeOH and F, DMSO. Random meander is black, β-sheet is red, β-bridge is white, bend is blue and turn is green.