Sequence environment of mutation affects stability and folding in collagen model peptides of osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI), a disorder characterized by fragile bones, is often a consequence of missense mutations in type I collagen, which change one Gly in the repeating (Gly-Xaa-Yaa)(n) sequence to a larger amino acid. The impact of local environment and the identity of the residue replacing Gly were investigated using two sets of triple-helical peptides. Gly mutations in the highly stable (Pro-Hyp-Gly)(10) system are compared with mutations in T1-865 peptides where the mutation is located within a less stable natural collagen sequence. Replacement of a Gly residue by Ala, Ser, or Arg leads to significant triple-helical destabilization in both peptide systems. The loss of stability (ΔT(m) ) due to a Gly to Ala or Gly to Ser change was greater in the more rigid (Pro-Hyp-Gly)(10) peptides than in the T1-865 set, as expected. But the final T(m) values, which may be the more biologically meaningful parameters, were higher for the (Pro-Hyp-Gly)(10) mutation peptides than for the corresponding T1-865 mutation peptides. In both peptide environments, a Gly to Arg replacement prevented the formation of a fully folded triple-helix. Monitoring of folding by differential scanning calorimetry showed a lower stability species as well as the fully folded triple-helical molecules for T1-865 peptides with Gly to Ala or Ser replacements, and this lower stability species disappears as a function of time. The difficulty in propagation through a mutation site in T1-865 peptides may relate to the delayed folding seen in OI collagens and indicates a dependence of folding mechanism on the local sequence environment.

Figure 1

[A] CD thermal unfolding profiles for (POG)₁₀ (black); the POG(G→A) peptide (A, red); the POG(G→S) peptide (S, cyan); and the POG(G→R) peptide (R, green). [B] CD thermal unfolding profiles for T1-865 (black); the T1-865(G→A) peptide (A, red); the T1-865(G→S) peptide (S, cyan); the T1-865(G→R) peptide (R, green); and the T1-865(G→D) (D, blue); [C] DSC transitions of (POG)₁₀, (POG)G→A, (POG)G→S, and (POG)G→R, using the same colors and notation described above. [D] DSC transitions of T1-865, T 1-865(G→A), T1-865(G→S), T1-865(G→R), and T1-865(G→D).

Figure 2

[A] CD folding curve of (POG)₁₀ (black), POG(G→A) (red), and POG(G→S) (cyan). [B] CD folding for T1-865 (black), T1-865(G→A) (red) and T1-865(G→S) (cyan).

Figure 3

[A] DSC profiles of POG(G→S) after refolding for 0.4hrs and 50 hrs; [B] DSC profiles of T1-865(G→S) 1mg/ml after folding for different lengths of time, 0.3, 1, 2, 5, 25 and 50 hrs (arrow represents increasing time); [C] DSC profiles of T1-865(G→S) 10 mg/ml after folding for periods of 0.3, 1, 2, 5, 25, and 50 hrs (arrow represents increasing time points); [D] Plot of Cp_max data for T1-865(G→S) taken from panels B and C as a function of time for the fully folded native state of 1mg/ml (solid squares) and 10mg/ml (open squares) and for the lower stability species transition at 1mg/ml (solid triangles) and 10mg/ml (open triangles).

Figure 4

Comparison of the impact of Gly substitutions on thermal stability (T_m) of the triple helix in different environments. On the left is illustrated the effect of a Gly to Ser replacement on the T_m value of (POG)₁₀ and T1-865, showing that the degree of destabilization ΔT_m is greater in the (POG)₁₀ context, yet the final stability T_m^mut is still higher for (POG)(G→S) than T1-865(G→S). On the right is illustrated the effect of a Gly _mut to Arg replacement, show a greater destabilization for (POG)₁₀, but very similar T_m final thermal stability for both peptide environments.

Figure 5

A) Scheme showing a linear model for folding of T1-865(G→S), nucleated at the C-terminal (POG)₄ sequence (red) and folding from the C- to N-terminus. A pause at the site of a Gly substitution could lead to a molecular species which is less stable than the fully folded molecule, which eventually leads to all fully folded species. (B) A more complex scheme where nucleation can be initiated largely or exclusively at the (POG)₄ C-terminus (red) at lower concentration, but which could be nucleated significantly at the N-terminus GPO(GAO)₃ sequence (blue) at higher concentrations. At high concentrations, it is possible that N-terminal nucleation would occur at significant amounts and lead to fully folded molecules at a faster rate than the C-terminal nucleation.