Characterization of collagen-like peptides containing interruptions in the repeating Gly-X-Y sequence

Abstract

Glycine is found as every third residue along the entire length of triple helices in fibrillar collagens, but the triple-helix regions of nonfibrillar collagens and other proteins usually contain one or more interruptions in this repeating pattern. A set of four peptides was designed to model the effect of interruptions in the (Gly-X-Y)n repeating pattern on triple-helix formation, stability, and folding. Into the middle of the stable triple-helical peptide (Pro-Hyp-Gly)10, an interruption was introduced representing one of the four possible categories: a glycine deletion, a deletion of a hydroxyproline (Y position), an alanine insertion, or a glycine to alanine substitution. As shown by sedimentation equilibrium, NMR, and CD studies, the introduction of an interruption still allowed formation of trimers in solution, but with marked decrease in stability. The degree of destabilization and the thermodynamic basis for the loss of stability depended on the type of interruption. The glycine substitution and alanine insertion were the least disruptive, followed by the hydroxyproline deletion, with the glycine deletion being the most destabilizing. Our results suggest that the breaks in these peptides affect both the triple-helical conformation and the monomer conformation. These studies provide a basis for considering the structural and functional consequences of different kinds of interruptions in collagen.

Acid destabilization of a triple‐helical peptide model of the macrophage scavenger receptor

“…With increasing temperature, the amplitude of this maximum undergoes a sharp decrease near 30°C (pH 7, 1 mg/ml). This sharp thermal transition, similar to that seen for trimer-to-monomer transitions of our other triple-helical peptides [10,17], suggests that MSR-1 adopts a triple-helical structure at temperatures below 30°C (Fig. 2).…”

“…Thermodynamic parameters were extracted by non-linear least squares fitting of the equilibrium constant for the transition between monomer and trimer as previously described [17]. After correcting for a linear temperature dependence in ellipticity for both monomer and trimer, the equilibrium melting transitions were fit to a two state trimer to monomer transition.…”

“…After correcting for a linear temperature dependence in ellipticity for both monomer and trimer, the equilibrium melting transitions were fit to a two state trimer to monomer transition. Thermodynamic parameters were calculated following previously described methods [17,18,19], using a standard state of 1 mM. The Tm and the van't Hoff enthalpy AH ° were determined by curve fitting to the following equation, where Co is the initial peptide concentration:…”

Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine

“…The host peptide GPO (19) as well as peptides GSO and GPS showed values of [⍜] 225 Ϸ 4,000 deg⅐cm 2 ⅐dmol Ϫ1 in native conditions, indicating a fully triple-helical conformation, as also found for other related host-guest peptides (19)(20)(21)(22)27). The corresponding values for peptides APO (3,600 deg⅐cm 2 ⅐dmol Ϫ1 ), CPO under oxidizing conditions (3,200 deg⅐cm 2 ⅐dmol Ϫ1 ), and SPO (3,200 deg⅐cm 2 ⅐dmol Ϫ1 ) are lower but are still in the range expected for a triple helix, whereas the CD amplitudes of peptides CPO red (1,600 deg⅐cm 2 ⅐dmol Ϫ1 ), DPO (2,000 deg⅐cm 2 ⅐dmol Ϫ1 ), EPO (1,700 deg⅐cm 2 ⅐dmol Ϫ1 ), RPO (1,600 deg⅐cm 2 ⅐dmol Ϫ1 ), and VPO (1,300 deg⅐cm 2 ⅐dmol Ϫ1 ) are comparable with the CD amplitude of a single-chain polyproline II helix (24,28,29).…”

“…Assuming a two-state mechanism in which three unfolded chains u combine to a triple-helical molecule n results in an equilibrium constant K ϭ c n ͞c u 3 ϭ F͞3 c 0 2 (1 Ϫ F) 3 where c 0 ϭ c u ϩ 3c n is the total peptide concentration and F ϭ 3c n ͞c 0 the fraction of folded peptide (23). This assumption seems justified, because a two-state transition was verified for related peptides by analytical ultracentrifugation (24).…”