Evaluation of Orientation-Dependent Cation-π Pairwise Effects within Collagen Triple Helices

Abstract

Various noncovalent interactions have been introduced to explore their impacts in folding a collagen triple helix. Among these interactions, the cation-π interaction represents one of the compelling forces stabilizing the triple helix. Still, the effects depend on the pairwise components and the orientation between the cationic and aromatic moieties. To gain more insights into this interaction within a collagen trimer, we prepared a series of collagen-mimetic peptides (CMPs) with cationic residues and aromatic residues incorporated to examine the contributions of two types of axial cation-π pairs (N → C and C → N cationic-to-aromatic pairwise) and the lateral cation-π pair. Circular dichroism (CD) measurements indicate that the N → C axial pairs have a significant stabilization effect. In contrast, the lateral and the C → N axial pairs destabilize the fold, and the lateral pairs cause the most destabilization consequences. We further designed and prepared the CMPs containing various lateral and axial cation-π pairs to investigate the coupling consequences in homotrimers and heterotrimers. From CD data, we found that the predicted differences in melting temperatures using individual cation-π pairwise contributions were comparable to the observed values for the designed homotrimers. CD and NMR measurements showed favorable cation-π interactions could effectively induce the folding of heterotrimers, in which the CMPs with more N → C axial pairs formed a more stable trimer than those containing a smaller number of N → C axial pairs. In this study, we have disclosed more valuable information about the properties of cation-π pairwise effects within a collagen triple helix, which can be considered in designing collagen-related peptides and materials.

Figure 1

Illustration of cation−π pairs with different orientations within a collagen triple helix. O is (2S,4R)-hydroxyproline and X is an aromatic residue. The boxes indicate the lateral pairs, the blue arrows indicate the N → C axial pairs, and the red arrows indicate the C → N axial pairs.

Figure 2

Illustration of cation−π pairs within a collagen homotrimer. The N → C axial pairs are shown in blue arrows, the C → N axial pairs in red arrows, and the lateral pairs in violet arrows. The bottom panel indicates the number of each pair in TRX3 and TXR3.

Figure 3

CD-monitored thermal unfolding transitions for TRX3 and TXR3 peptides. All the measurements were conducted in pH 7.0 and 20 mM phosphate buffer with a peptide concentration of 0.2 mM. The heating rate for the measurements was 0.16 °C/min. The solid lines represent the best fit for curves using a two-state model.

Figure 4

DSC thermograms for the triple helices derived from TRX3 and TXR3 peptides. All the measurements were conducted in pH 7.0 and 20 mM phosphate buffer with a peptide concentration of 0.6 mM. The heating rate for the measurements was 0.1 °C/min. The solid lines represent the best fit for the experimental data (dashed lines).

Figure 5

CD-monitored thermal unfolding transitions for Ca, Cb, and their mixtures. All the measurements were conducted in pH 7.4 and 20 mM phosphate buffer with a peptide concentration of 0.2 mM. The heating rate for the measurements was 0.16 °C/min. The solid lines represent the best fit for curves using a two-state model.

Figure 6

Overlapped ¹H, ¹⁵N–HSQC spectra of 2Ca/1Cb (violet) and 1Ca/2Cb (magenta) at 10 °C. The (t) denotes trimers for the heterotrimers. Ca* and Cb* peptides were used in the measurements.