Missense mutations in the collagen triple helix that replace one Gly residue in the (Gly-X-Y) n repeating pattern by a larger amino acid have been shown to delay triple helix folding. One hypothesis is that such mutations interfere with the C-to N-terminal directional propagation and that the identity of the residues immediately N-terminal to the mutation site may determine the delay time and the degree of clinical severity. Model peptides are designed to clarify the role of tripeptide sequences N-terminal to the mutation site, with respect to length, stability, and nucleation propensity, to complete triple helix folding. Two sets of peptides with different N-terminal sequences, one with the natural sequence ␣1(I) 886 -900, which is just adjacent to the Gly 901 mutation, and one with a GPO(GAO) 3 sequence, which occurs at ␣1(I) 865-879, are studied by CD and NMR. Placement of the five tripeptides of the natural ␣1(I) collagen sequence N-terminal to the Gly to Ala mutation site results in a peptide that is folded only C-terminal to the mutation site. In contrast, the presence of the Hyp-rich sequence GPO(GAO) 3 N-terminal to the mutation allows complete refolding in the presence of the mutation. The completely folded peptide contains an ordered central region with unusual hydrogen bonding while maintaining standard triple helix structure at the N-and C-terminal ends. These peptide results suggest that the location and sequences of downstream regions favorable for renucleation could be the key factor in the completion of a triple helix N-terminal to a mutation.Abnormalities in protein folding are known to play a role in many diseases, including those arising from mutations in the collagen triple helix (1). The best characterized collagen disease is osteogenesis imperfecta (OI), 5 or brittle bone disease, in which there is defective mineralization of bones in type I collagen (2, 3). Missense mutations that change one Gly in the repetitive (Gly-X-Y) sequence are the most common mutations (4). Such Gly mutations are found all along the collagen chain, suggesting that the loss of a Gly at any site in the triple helix has pathological consequences. The phenotype of the disease varies widely, depending on the type of amino acid substitution and the site of the mutation (5, 6). There is evidence of abnormal folding of collagen in OI and other collagen diseases, which may relate to the pathology (7, 8).Folding of the triple helix is a complex, multistep process that includes association of three chains to form the supercoiled polyproline II triple helical structure (8). Collagen is synthesized in a procollagen form, with N-and C-terminal globular propeptides flanking the (Gly-X-Y) n central domain (9, 10). Posttranslational hydroxylation of Pro and Lys residues in the Y positions and further glycosylation of Hyl occur while the chains are unfolded (11, 12). Trimerization occurs through the association of three C-terminal propeptides, and then nucleation of the triple helix takes place at the (Gly-Pro-Hyp) n -rich sequence...

Protein folding is determined by molecular features in the unfolded state, as well as the native folded structure. In the unfolded state, imino acids both restrict conformational space and present cis-trans isomerization barriers to folding. Because of its high proline and hydroxyproline content, the collagen triple-helix offers an opportunity to characterize the impact of imino acids on the unfolded state and folding kinetics. Here, NMR and CD spectroscopy are used to characterize the role of imino acids in a triple-helical peptide, T1-892, which contains an 18-residue sequence from type I collagen and a C-terminal (Gly-Pro-Hyp)(4) domain. The replacement of Pro or Hyp by an Ala in the (Gly-Pro-Hyp)(4) region significantly decreases the folding rate at low but not high concentrations, consistent with less efficient nucleation. To understand the molecular basis of the decreased folding rate, changes in the unfolded as well as the folded states of the peptides were characterized. While the trimer states of the peptides are all similar, NMR dynamics studies show monomers with all trans (Gly-Pro-Hyp)(4) are less flexible than monomers containing Pro --> Ala or Hyp --> Ala substitutions. Nucleation requires all trans bonds in the (Gly-Pro-Hyp)(4) domain and the constrained monomer state of the all trans nucleation domain in T1-892 increases its competency to initiate triple-helix formation and illustrates the impact of the unfolded state on folding kinetics.