Template-tethered collagen mimetic peptides for studying heterotrimeric triple-helical interactions

Abstract

Collagen mimetic peptides (CMPs) have been used to elucidate the structure and stability of the triple helical conformation of collagen molecules. Although CMP homotrimers have been widely studied, very little work has been reported regarding CMP heterotrimers because of synthetic difficulties. Here, we present the synthesis and characterization of homotrimers and ABB type heterotrimers comprising natural and synthetic CMP sequences that are covalently tethered to a template, a tris(2-aminoethyl) amine (TREN) succinic acid derivative. Various tethered heterotrimers comprising synthetic CMPs [(ProHypGly)₆, (ProProGly)₆] and CMPs representing specific domains of type I collagen were synthesized and characterized in terms of triple helical structure, thermal melting behavior, and refolding kinetics. The results indicated that CMPs derived from natural type I collagen sequence can form stable heterotrimeric helical complexes with artificial CMPs and that the thermal stability and the folding rate increase with the increasing number of helical stabilizing amino acids (e.g. Hyp) in the peptide chains. Covalent tethering enhanced the thermal stability and refolding kinetics of all CMPs; however, their relative values were not affected suggesting that the tethered system can be used for comparative study of heterotrimeric CMP's folding behavior in regards to chain composition and for characterization of thermally unstable CMPs.

Figure 1

Thermal melting curves of untethered peptides O, T, P and α.

Figure 2

Thermal melting curves and CD spectra (inset) of untethered peptide P and template-tethered P·P·P. The high ellipticity at 226 nm and the sigmoidal melting curves for P·P·P indicate triple helix stabilization effect of the TREN template.

Figure 3

CD melting studies of peptide O, and template-tethered CMP homotrimers and heterotrimers composed of P and O.

Figure 4

Refolding kinetics of thermally denatured template-tethered CMP homotrimers and heterotrimers at 4°C.

Figure 5

CD spectra (a) and thermal melting curves (b) of α and α-containing template-tethered homotrimers and heterotrimers, shown as mean residue ellipticity. (c) Thermal melting curves of heterotrimer α·P·P and α·O·O, shown as fraction folded. Peptide α and homotrimer α·α·α do not self assemble into triple helices, but heterotrimers α·P·P and α·O·O form stable triple helices with defined T_m values.

Figure 5

CD spectra (a) and thermal melting curves (b) of α and α-containing template-tethered homotrimers and heterotrimers, shown as mean residue ellipticity. (c) Thermal melting curves of heterotrimer α·P·P and α·O·O, shown as fraction folded. Peptide α and homotrimer α·α·α do not self assemble into triple helices, but heterotrimers α·P·P and α·O·O form stable triple helices with defined T_m values.

Figure 5

CD spectra (a) and thermal melting curves (b) of α and α-containing template-tethered homotrimers and heterotrimers, shown as mean residue ellipticity. (c) Thermal melting curves of heterotrimer α·P·P and α·O·O, shown as fraction folded. Peptide α and homotrimer α·α·α do not self assemble into triple helices, but heterotrimers α·P·P and α·O·O form stable triple helices with defined T_m values.

Figure 6

Thermal melting curves of T·T·T, T·P·P and T·O·O, shown as mean residue ellipticity (a), and fraction folded (b).

Figure 6

Thermal melting curves of T·T·T, T·P·P and T·O·O, shown as mean residue ellipticity (a), and fraction folded (b).

Figure 7

CD spectra (a) and thermal melting curves (b) of α·O·O under acidic, basic and neutral conditions.

Figure 7

CD spectra (a) and thermal melting curves (b) of α·O·O under acidic, basic and neutral conditions.

Scheme 1

Synthetic strategy for template-tethered ABB type CMP heterotrimers.