Conformational dynamics and stability of HP35 studied with 2D IR vibrational echoes

Abstract

Two-dimensional infrared (2D IR) vibrational echo spectroscopy was used to measure the fast dynamics of two variants of chicken villin headpiece 35 (HP35). The CN of cyanophenylalanine residues inserted in the hydrophobic core were used as a vibrational probe. Experiments were performed on both singly (HP35-P) and doubly CN-labeled peptide (HP35-P(2)) within the wild-type sequence, as well as on HP-35 containing a singly labeled cyanophenylalanine and two norleucine mutations (HP35-P NleNle). There is a remarkable similarity between the dynamics measured in singly and doubly CN-labeled HP35, demonstrating that the presence of an additional CN vibrational probe does not significantly alter the dynamics of the small peptide. The substitution of two lysine residues by norleucines markedly improves the stability of HP35 by replacing charged with nonpolar residues, stabilizing the hydrophobic core. The results of the 2D IR experiments reveal that the dynamics of HP35-P are significantly faster than those of HP35-P NleNle. These observations suggest that the slower structural fluctuations in the hydrophobic core, indicating a more tightly structured core, may be an important contributing factor to HP35-P NleNle's increased stability.

Figure 1

Structural representation of HP35-P. Phe58 has a CN functional group that serves as the vibrational probe. Two other phenylalanine residues participating in the aromatic stacking in the hydrophobic core are shown. Lys65 and Lys70 are highlighted in orange.

Figure 2

FT IR spectra of the CN stretching mode of HP35-P (blue) and HP35-P₂ (black). The inset shows the spectrum prior to the background subtraction of a large water absorption band. Within experimental error the two spectra are identical.

Figure 3

CLS data for the CN stretching mode of HP35-P (blue) and HP35-P₂ (black). Within experimental error the data are identical, which demonstrates that the addition of a second CN vibrational dynamics label (HP35-P₂) does not change the structural fluctuations of the peptide.

Figure 4

The unfolding of HP35-P (blue circles) and HP35-P NleNle (green triangles) measured with CD signal at 222 nm. The results show the increased stability of HP35-P NleNle relative to HP35-P, which has been reported previously for NNHP35 and HP35.

Figure 5

FT IR spectra of the CN stretching mode in HP35-P (blue) and HP35-P NleNle (green). The center frequencies are 2233.8 ± 0.2 and 2233.8 ± 0.2 cm^-1 for HP35-P and HP35-P NleNle, respectively. The full width at half maximum (FWHM) is 13.5 ± 0.2 cm^-1 for both peptides.

Figure 6

2D IR vibrational echo spectra of the CN in HP35-P (top row) and HP35-P NleNle (bottom row). T_w = 0.4 ps (left panels) and T_w = 4.2 ps (right panels) are shown.

Figure 7

The CLS decay data for HP35-P (blue circles) and HP35-P NleNle (green triangles). The data show clearly that the structural dynamics of the mutant HP35-P NleNle are significantly slower than those of HP35-P.