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.
Highlights
A paradigm for how Pro and Hyp stabilize the triple helix has emerged recently [5,6,7,8,9,10,11,12,13]
Pro derivatives that prefer the C␥-endo pucker stabilize the triple helix when substituted in the Xaa position, whereas Pro derivatives that prefer the C␥-exo pucker stabilize the triple helix when substituted in the Yaa position [1, 14]
We began our analysis by testing the possibility that the hyperstability of (HypHypGly)n triple helices results merely from a Pro derivative with a high PPII propensity and Ktrans/cis ratio being in the Xaa and Yaa positions simultaneously
Summary
Peptide Synthesis—The peptides (GlyFlpFlp), (GlyFlpHyp), (GlyHypFlp), (GlyProFlp), and (HypMepGly) were synthesized by segment condensation of Fmoc-GlyFlpFlp-OH, FmocGlyFlpHyp-OH, Fmoc-GlyHypFlp-OH, Fmoc-GlyProFlp-OH, and Fmoc-Hyp(tBu)MepGly-OH tripeptides, respectively. Differential Scanning Calorimetry—Differential scanning calorimetry (DSC) measurements on peptides (GlyHypFlp), (GlyProFlp), (GlyFlpHyp), (GlyHypHyp), (GlyProHyp), and (HypMepGly) were conducted on a VP-DSC instrument from MicroCal (Northampton, MA). After DSC measurements, peptide concentrations were determined by quantitative amino acid analysis (Protein Chemistry Core, Biomolecular Resource Facility, University of Texas Medical Branch). Peptide concentrations of 138, 136, 130, 289, 176, and 478 M were discerned for (GlyFlpHyp), (GlyHypFlp), (GlyProFlp), (HypMepGly), (GlyProHyp), and (GlyHypHyp), respectively. The value of ⌬S at the Tm was calculated by using the equation: Tm ϭ ⌬H/(⌬S ϩ R1⁄7ln(0.75c2)), where c is the concentration of monomeric peptide determined by amino acid analysis and Tm is the maximum of the DSC endotherm [44, 45]. A small baseline of A Յ 0.1 at 230 nm was employed at both temperatures for each peptide, but improved the fit only slightly and did not alter any conclusions
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