Interstrand dipole-dipole interactions can stabilize the collagen triple helix

Abstract

The amino acid sequence of collagen is composed of GlyXaaYaa repeats. A prevailing paradigm maintains that stable collagen triple helices form when (2S)-proline (Pro) or Pro derivatives that prefer the C(γ)-endo ring pucker are in the Xaa position and Pro derivatives that prefer the C(γ)-exo ring pucker are in the Yaa position. Anomalously, an amino acid sequence in an invertebrate collagen has (2S,4R)-4-hydroxyproline (Hyp), a C(γ)-exo-puckered Pro derivative, in the Xaa position. In certain contexts, triple helices with Hyp in the Xaa position are now known to be hyperstable. Most intriguingly, the sequence (GlyHypHyp)(n) forms a more stable triple helix than does the sequence (GlyProHyp)(n). Competing theories exist for the physicochemical basis of the hyperstability of (GlyHypHyp)(n) triple helices. By synthesizing and analyzing triple helices with different C(γ)-exo-puckered proline derivatives in the Xaa and Yaa positions, we conclude that interstrand dipole-dipole interactions are the primary determinant of their additional stability. These findings provide a new framework for understanding collagen stability.

FIGURE 1.

Preferred ring conformations of Pro and Pro derivatives. The C^γ-exo conformation is favored strongly by the gauche effect when R¹ = OH (Hyp) or F (Flp), R² = H, and by steric effects when R¹ = H, R² = CH₃ (Mep). The C^γ-endo:C^γ-exo ratio is ∼2 when R¹ = R² = H (Pro) (13).

FIGURE 2.

Conformational analysis of peptides. A–C, circular dichroism spectra of peptide solutions (∼0.2 mm in 50 mm sodium phosphate buffer, pH 7.0; except (GlyFlpFlp)₇, which was ∼0.01 mm) at 4 °C after incubating at 4 °C for ≥24 h. D–F, effect of temperature on the molar ellipticity at 225 nm. Data were recorded at 3-°C intervals after a 5-min equilibration.

FIGURE 3.

Sedimentation equilibrium analysis of peptides. Circles, 4 °C; squares, 37 or 40 °C. For clarity, only every third data point is shown. Best fits are shown as solid or dashed lines. A, (HypMepGly)₇ (40,000 rpm at 4 °C and 50,000 rpm at 37 °C). Fits shown are for monomer at both 4 and 37 °C. B, (GlyFlpHyp)₇ (45,000 rpm at 4 °C and 42,000 rpm at 40 °C) and (GlyHypHyp)₇ (40,000 rpm at 4 °C and 50,000 rpm at 37 °C). Fits shown are for trimer at 4 °C and monomer at 37 or 40 °C. C, (GlyHypFlp)₇ (45,000 rpm at 4 °C and 42,000 rpm at 40 °C) and (GlyProFlp)₇ (45,000 rpm at 4 °C and 42,000 rpm at 40 °C). Fits shown are for trimer at 4 °C, and a mixture of monomer and trimer at 40 °C.

FIGURE 4.

Differential scanning calorimetry of peptides. A, (HypMepGly)₇ (289 μm). B, (GlyHypHyp)₇ (478 μm), (GlyFlpHyp)₇ (138 μm), and (GlyProHyp)₇ (176 μm). C, (GlyProFlp)₇ (130 μm) and (GlyHypFlp)₇ (136 μm). Data were collected with a scan rate of 6 °C/h.

FIGURE 5.

Illustration of dipoles in the side chains of proximal Xaa and Yaa residues in a (GlyXaaYaa)_n triple helix. Images were created by modifying Hyp residues in panel A, which shows a cross section of a (HypHypGly)₁₀ triple helix (32), with the program PyMOL (Delano Scientific, Palo Alto, CA). The dipole-dipole interaction in panel A could contribute 0.6 kcal/mol to the stability of the triple helix (37), and likely more in panel B.