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.
Triple-helix formation of the peptide (Pro-Hyp-Gly)10 was monitored by nmr and CD spectroscopy. The two-dimensional nmr spectra indicated that the Gly C alpha H and Pro C delta H proton resonances shift upfield in going from the nonhelical to helical form, while hydroxy-proline resonances are unchanged. The integrated areas of the helical and nonhelical resonances could be monitored in the one-dimensional nmr spectrum, and indicate that in the (Pro-Hyp-Gly)10 about 90% of the residues are in a defined triple-helical conformation. The introduction of a glycine to alanine substitution or the deletion of a single hydroxyproline residue in the stable triple-helical peptide (Pro-Hyp-Gly)10 still allows trimers to be formed, but the trimers show a substantial loss of triple helix and decreased thermal stability compared with (Pro-Hyp-Gly)10. Two computer models were generated for the Gly----Ala peptide, one with the Ala side chains packed inside the helix and the other with the region containing the alanines forming a beta-bend that loops out from the helix. The nmr data is more consistent with the latter model.
The triple-helical domains of type IV collagen chains have more than 20 sites at which the repeating (Gly-X-Y)n pattern is interrupted. Analysis of alpha 1 (IV) and alpha 2 (IV) chains indicates the residues in the three Gly-X-Y triplets preceding or following interruptions differ statistically from the rest of the chain. Unusually high frequencies of charged residues are seen at a number of X and Y sites, with the charge density being particularly high C-terminal to the interruption site. Analyses were carried out on individual categories of interruptions, classified as insertions or deletions in the Y position. All of the residues in the X and Y positions of the triplets flanking insertion sites are atypical, with a high concentration of charged residues. Triplets flanking sites where there has been a deletion in the Y position show unusually high frequencies of charged residues at some sites, hydrophobic residues at other sites, and an invariant imino acid N-terminal to the interruption. The presence of atypical sequences surrounding interruptions could be important at a molecular level, related to triple-helix stability, or at a supramolecular level, related to the association of molecules to form networks in basement membranes.
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