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

Abstract

Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa)(n) triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host-guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp)(3)-Zaa-Pro-Hyp-(Gly-Pro-Hyp)(4)-Gly-Gly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa = Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa = Gly (melting temperature, T(m) = 45 degrees C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T(m) decreases to approximately 10 degrees C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, T(m) < 0 degrees C. A Gly --> Cys replacement results in T(m) < 0 degrees C under reducing conditions but shows a broad transition (T(m) approximately 19 degrees C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T(m) by approximately 5 degrees C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala </= Ser < CPO(red) < Arg < Val < Glu </= Asp. The rank of destabilization of substitutions for Gly in these Gly-Pro-Hyp-rich homotrimeric peptides shows a significant correlation with the severity of natural OI mutations in the alpha1 chain of type I collagen.

Figure 1

Far-UV CD spectra of host–guest peptides. CPO_red is used to indicate the peptide with a Cys-Pro-Hyp guest triplet under reducing conditions. (A) Spectra are shown for peptides GPS (—), SPO (----), CPO_red (-⋅⋅-⋅⋅), DPO (⋅⋅⋅⋅⋅⋅⋅⋅), and RPO (-⋅-⋅-) recorded at 0°C in PBS (concentration = 1 mg/ml). Corresponding spectra are shown for peptides GSO (B) and APO (C) recorded at TMAO concentrations of (from bottom to top at 225 nm) 0, 0.5, 1.0, 1.5, 2.0, and 3.7 M. (Insets) The increase of the molar ellipticity at 225 nm observed on increasing the concentrations of TMAO.

Figure 2

Thermal denaturation of host–guest peptides. The change of ellipticity on heating was monitored at 225 nm for peptides APO (—), SPO (----), CPO_red (-⋅⋅-⋅⋅), RPO (⋅⋅⋅⋅⋅⋅⋅⋅), and DPO (-⋅-⋅-⋅) (concentration = 1 mg/ml) in PBS in the absence (A) and presence (B) of 3.7 M TMAO. Transition curves were normalized to the fraction of folded peptides for profiles in the absence (C) and presence (D) of TMAO. Transition profiles for peptides VPO (short dotted line) and EPO (short dashed line) are included in D.

Figure 3

Thermal denaturation of host–guest peptides at varying concentrations of TMAO. The change of ellipticity on heating was monitored at 225 nm for peptides GSO (A) and APO (B) (concentration = 1 mg/ml) in PBS (—) and after addition of 0.5 (----), 1.0 (⋅⋅⋅⋅⋅⋅⋅⋅), 2.0 (-⋅-⋅-⋅), and (for APO) 3.7 (-⋅⋅-⋅⋅) M TMAO. (Insets) The increase in melting temperature on increasing TMAO concentrations.

Figure 4

Distribution of Gly → Xaa mutations within the collagen type I triple-helical region causing OI. Residues replacing Gly within the Gly-Xaa-Yaa triplets are indicated (●, Ala; ♦, Arg; *, Asp; ○, Cys; □, Glu; ▴, Ser; ▵, Val) and classified as mild, moderate, or severe. Mutations are those collected in the collagen mutation database (www.le.ac.uk/genetics/collagen/; ref. 10), and further unpublished ones are included with permission of the group of A. De Paepe (personal communication). Dotted lines indicate positions for which different mutations are known where the severity of the disease depends on the identity of the encoded amino acid residue.